Edible foodstuffs and bio reactor design

ABSTRACT

Methods of production of edible filamentous fungal biomat formulations are provided as standalone protein sources and/or protein ingredients in foodstuffs as well as a one-time use or repeated use self-contained biofilm-biomat reactor comprising a container with at least one compartment and placed within the compartment(s), a feedstock, a fungal inoculum, a gas-permeable membrane, and optionally a liquid nutrient medium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/116,836, filed 29 Aug. 2018, which claims the benefit of priority ofU.S. Provisional Patent Applications 62/722,074, filed 23 Aug. 2018, and62/552,093, filed 30 Aug. 2017. The entireties of all of theabove-referenced applications are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Small BusinessTechnology Transfer (STTR) program grant numbers STTR NASA 80NSSC20C0031and STTR NASA 80NSSC18P2141 awarded by the National Aeronautics andSpace Administration. The government has certain rights in theinvention.

TECHNICAL FIELD

This application relates to edible fungi and provides methods ofpreparing edible fungi for use in foodstuffs, liquid and solidformulations of edible fungi, as well as uses and methods associatedtherewith, foodstuffs containing edible fungi, and methods and usesthereof.

BACKGROUND

The United Nations listed the world population as 7.5 billion in August2017 and predicts that figure to grow to 8 billion in 2023 and to be 10billion in 2056. In a related report, the Food and AgriculturalOrganization of the United Nations (FAO) estimates that if the globalpopulation reaches 9.1 billion by 2050, world food production will needto rise by 70% and to double in the developing world. That increase infood production will need to occur despite rising energy costs,decreasing underground aquifer resources, loss of farm land to urbansprawl, and increasingly severe weather due to climate change (e.g.increased temperatures, increased drought, increased flooding, etc.).This is a particular challenge for countries such as Africa which,according to 2009 figures, already has inadequate protein intake andcountries such as China, India, Pakistan, and Indonesia which are atrisk of inadequate protein intake. In addition, the global demand isforecasted for 2040 to increase by 60% for meat and 50% for dairy.

But not all protein sources are created equal. Animal based foods (meat,eggs, dairy) provide “complete” proteins as they contain all of theessential amino acids; that is, methionine, leucine, isoleucine,phenylalanine, valine, threonine, histidine, tryptophan and lysine.Plant based foods, while containing some essential amino acids,generally lack the complete set. For example, the protein found instarchy roots lacks the essential amino acid lysine, which must then beobtained from another food in the diet. Beans and legumes contain highlevels of lysine, but they lack the essential amino acid methionine.Although it is possible to build a complete protein by pairing plantfoods, ensuring a nutritionally balanced diet is much easier withcomplete proteins.

One non-animal source of a complete protein is obtained from ediblefilamentous fungi, such as Fusarium venenatum (formerly classified andFusarium graminearum). However, to date protein production from thesesources has required significant investment in energy resources andproduction equipment, such as capital-intensive bioreactors andcentrifuges. There remains a need for growth, harvesting, and foodstuffproduction methods that require low energy, consume few naturalresources, and are low cost. The current invention solves theseproblems.

In addition, one area of reducing the logistics supply associated withresponding to natural disasters, logistically isolated environments ormilitary and/or space/extraterrestrial missions is the closure of lifesupport loops, particularly waste streams, while providing missioncritical products such as nutritional and appetizing foods, fuels,metabolite expression platforms, building materials and/or microbialfactories. Oftentimes these types of environments have no or limitedaccess to sterile facilities and/or require a sealed aseptic system tofully contain the waste stream and/or food, fuel and materials produced.For example, work by the European Space Agency (Expeditions 25-28,Growth and Survival of Colored Fungi in Space (CFS-A)) demonstrated thatfungi can grow inside the space station and could decompose food andother organic materials in humid conditions; here containment of thefungal system is paramount to preventing inadvertent contamination ofother supplies and surfaces. In addition to the need to decompose foodand waste in the developing area of space travel, these needs are alsopresent when dealing with natural disasters, in-theater militaryoperations, wilderness operations, situations in the third world wheresanitation and refrigeration are not reliable, confined spaces,logistically difficult arenas and in some agricultural/industrialoperations. Having a self-contained aseptic system that operatesefficiently with a minimum of space, energy, and maintenance is needed.

A robust and efficient portable self-contained biofilm-biomat reactorsystem that is able to convert a wide variety of waste streams into amultitude of valuable products addresses these problems. The currentdisclosure describes a simple aseptic bioreactor platform that requiresno agitation, no active aeration, no energy source during fermentation(other than temperature control), generates minimal to no wasteresidues, requires little water, and produces dense, easily harvested,textured biomats. In addition, the self-contained biofilm-biomat reactorsystem can be portable and/or scalable for larger, more concentratedmissions and/or populations.

SUMMARY

The present disclosure provides formulations of edible filamentousfungi. The edible filamentous fungi are grown on liquid media undersurface fermentation conditions to produce filamentous fungal biomats.In one embodiment, a method for surface fermentation production ofedible fungal biomats is provided, the method comprising inoculating aliquid synthetic growth media containing a carbon source with planktonicand/or microconidial fungal cells, incubating the inoculated growthmedia at room temperature and harvesting a cohesive biomat produced bythe fungus. In some embodiments the inoculated growth media is incubatedin open trays or in open trays contained in at least a semi-sterileenvironment

In another embodiment, the production method for surface fermentationedible fungal biomat production allows harvesting a section of thebiomat while maintaining the growth potential of the remaining biomat.

In a further embodiment, the filamentous fungus is Fusarium oxysporumstrain MK7 (ATCC PTA-10698 deposited with the American Type CultureCollection, 1081 University Boulevard, Manassas, Va., USA), which has46-51% complete protein content with high levels of all essential aminoacids, specifically, 42-43% essential amino acids and 4-21% BCAA, whichis higher than eggs. In addition, Fusarium oxysporum strain MK7 has a8-10% minerals and ash content, including high levels of Calcium (1.3mg/100 g serving), Iron (5.5 mg/100 g serving), 1-2% nucleic acid, and6-11% lipid, of which 85% is unsaturated.

In another embodiment, the filamentous fungus is Fusarium venenatum orFusarium fujikuroi.

In still another embodiment, the filamentous fungus is selected from thegroup consisting of Agaricus bisporus (crimini and white), Boletusedulis (porcinini), Cantarellus cibarius (chantrelle), Calvatia gigantea(giant puffball), Cyclocybe aegerita (velvet piopinni), Ganodermalucidum (Reishi), Grifola frondosa (maitake), Morchella species (Morel),Hypsizygus tessellatus (clamshell), Hypsizygus ulmarius (elm oyster),Laetiporus species (chicken of the woods), Lentinula edodes (shiitake),Pleurotus eryngii (trumpet royale, king oyster), Calvatia gigantean(giant puffball), Pleurotus ostreatus (pearl oyster), Pleurotusostreatus var. columbinus (blue oyster) and other Pleurotus sp. (e.g.,P. citrinopileatus, tuberregium), Hypsizygus ulmarius (elm oyster),Pholiota microspora (forest nameko), Sparassis crispa (cauliflower), andTuber species (truffles).

In an additional embodiment, the carbon source is a sugar (e.g. sucrose,maltose, glucose, fructose, rare sugars, etc.), a sugar alcohol (e.g.glycerol, polyol, etc.), a starch (e.g. corn starch, etc.), a starchderivative, a starch hydrolysate, a hydrogenated starch hydrolysate, alignocellulosic pulp or feedstock (e.g. sugar beet pulp, agriculturalpulp, lumber pulp, distiller dry grains, brewery waste, etc.), cornsteep liquor, acid whey, sweet whey, milk serum, wheat steep liquor,industrial liquor, food refinery products/waste streams, and/orcombinations thereof.

In yet another embodiment, a method for surface fermentation productionof edible filamentous fungal biomats initiated from the fruiting bodiesor spores of filamentous fungi is provided. For biomats initiated fromfruiting bodies, the method comprises surface sterilizing the fruitingbody of the fungus, reducing the size of the sterilized fruiting body ofthe fungus, surface sterilizing the reduced fruiting body of the fungus,inoculating a synthetic liquid growth media containing a carbon sourcewith cells from the sterilized reduced fruiting body of the fungus,incubating the inoculated growth media at room temperature, andharvesting a cohesive filamentous biomat produced by the fungus. Forbiomats initiated from filamentous fungal spores, the method comprisesinoculating a synthetic liquid growth media containing a carbon sourcewith sterile spores, incubating the inoculated growth media at roomtemperature, and harvesting a cohesive filamentous biomat produced bythe fungal spores.

In some embodiments, the fruiting body or spores of the filamentousfungus is selected from the group consisting of Agaricus bisporus(crimini and white), Boletus edulis (porcinini), Cantarellus cibarius(chantrelle), Calvatia gigantea (giant puffball), Cyclocybe aegerita(velvet piopinni), Ganoderma lucidum (Reishi), Grifola frondosa(maitake), Morchella species (Morel), Hypsizygus tessellatus(clamshell), Hypsizygus ulmarius (elm oyster), Laetiporus species(chicken of the woods), Lentinula edodes (shiitake), Pleurotus eryngii(trumpet royale), Pleurotus ostreatus (pearl oyster and blue oyster),Pholiota microspora (forest nameko), Sparassis crispa (cauliflower), andTuber species (truffles). Sterile spores of the filamentous fungi wereobtained from commercial venders, such as Myco Direct (Huntley, Ill.).

In still another embodiment, the filamentous biomat produced fromplanktonic cells, microconidia cells, sized reduced fruiting body, orspores of a filamentous fungus comprises less than 5 mm long aggregatesof mycelia and/or hyphae. In yet another embodiment, the size reducedfilamentous biomat comprises aggregates that are greater than 5 mm long.

In a further embodiment, the pH of the fruiting body cell inoculatedgrowth media has a pH of about 4.0-4.1.

In another further embodiment, the carbon source for the syntheticgrowth media for fruiting body and/or cell growth comprises glycerol,starch, corn steep liquor, acid whey or combinations thereof and/or theincubation period is about 2-10 days or longer.

Another embodiment relates to a formulation of edible fungus filamentousbiomat comprising edible fungal filamentous biomat particles isolatedfrom the edible fungus filamentous biomats grown via surfacefermentation on a synthetic liquid media.

Further embodiments relate to formulations that are in the form of aliquid, a solid or a gel.

Yet more embodiments relate to a formulation that is a paste, a flour, aporous/aerated mass and/or a firm mass.

Still another embodiment relates to a foodstuff comprising theformulation(s) of edible fungus filamentous biomat with or without otheringredients.

Additional embodiments are directed to foodstuffs made from theformulation(s) such as meat substitutes, drinks, beverages, yogurt,dessert, confections, or candy.

Another embodiment relates to a foodstuff made from the formulation(s)that is a mouse or a frozen dessert, such as an ice cream analogue, thatdoes not melt at room temperatures.

Further embodiments relate to the use of the formulation(s) as aningredient to augment and/or simulate the texture of a meat (e.g. aburger, sausage, hot dog, chicken or turkey nugget, and/or fish filet)in a foodstuff and/or to increase protein content of the foodstuff. Yetfurther embodiments relate to the use of liquid dispersionformulation(s) as a milk substitute and/or to increase the proteincontent of milk, milk products and/or milk substitute products.

Yet another embodiment relates to isolation of oils from an ediblefilamentous fungal biomat.

The present disclosure also provides a self-contained biofilm-biomatreactor. In one embodiment, the self-contained biofilm-biomat reactorcomprises a container and placed within the container a feedstock, afungal inoculum, a gas-permeable membrane(s), and optionally a liquidnutrient medium. In some embodiments the reactor is a one-time usereactor while in other embodiments the reactor can be reused.

Typically, the container in the various embodiments is capable of beingsealed and may include a container cover in addition to a seal. In someembodiments the container is a covered tray. In other embodiments thecontainer is a covered petrie dish or other type of covered container.In yet other embodiments, the container is a bag. In yet otherembodiments, the container is a pipe with the upper portion made of agas permeable membrane (2) (see FIG. 23). In some embodiments thecontainer is comprised of a plurality of growth compartments. In someembodiments the container has a manifold design and/or a bafflingsystem. In some embodiments the container is produced, either fully orpartially, from one or more consumable feedstocks.

In some embodiments the feedstock is inoculated with an ascomycetesfungal strain, such as Fusarium, examples of which are Fusariumoxysporum strain MK7 (ATCC PTA-10698 deposited with the American TypeCulture Collection, 1081 University Boulevard, Manassas, Va., USA),Fusarium venenatum, and Fusarium avenaceum, Fusarium fujikuroi, Rhizopusspecies, Aspergillus species, and/or combinations thereof.

In other embodiments the feedstock is inoculated with a basidiomycetesfungal strain, such as Agaricus bisporus (crimini and white), Boletusedulis (porcinini), Cantarellus cibarius (chantrelle), Calvatia gigantea(giant puffball), Cyclocybe aegerita (velvet piopinni), Ganodermalucidum (Reishi), Grifola frondosa (maitake), Morchella species (Morel),Hypsizygus tessellatus (clamshell), Hypsizygus ulmarius (elm oyster),Laetiporus species (chicken of the woods), Lentinula edodes (shiitake),Pleurotus eryngii (trumpet royale), Pleurotus ostreatus (pearl oysterand blue oyster), Pholiota microspora (forest nameko), Sparassis crispa(cauliflower), and/or Tuber species (truffles).

In some embodiments the feedstock is a waste product, such as naturallyoccurring urine and/or feces, as well as food waste and by-products,industrial waste and/or by-products, agricultural waste and by-products,plant material, and/or combinations thereof. In other embodiments thefeedstock can be a synthesized or manufactured surrogate, such assurrogate human urine. With respect to feedstock that is or includesplant material, that plant material is typically lignocellulosic. Thelignocellulosic feedstock is selected from the group consisting ofagricultural crop residues (e.g. wheat straw, barley straw, rice straw,pea, oat, small grain straw, corn stover, corn fibers (e.g. corn fibergum (CFG), distillers dried grains (DDG), corn gluten meal (CGM), switchgrass, hay-alfalfa, sugarcane bagasse, non-agricultural biomass (e.g.algal biomass, cyanobacterial biomass, urban tree residue), vegetables(e.g. carrots, broccoli, garlic, potato, beets, cauliflower), forestproducts and industry residues (e.g., softwood first/secondary millresidue, hard softwood first/secondary mill residue, recycled paper pulpsludge, anaerobic digestate), lignocellulosic containing waste (e.g.newsprint, waste paper, brewing grains, used rubber tire (URT),municipal organic waste, yard waste, clinical organic waste, sugar,starch, waste oils, olive oils, olive oil processing waste, cricketexcrement, and waste generated during the production of biofuels (e.g.processed algal biomass, glycerol), and combinations thereof. Typically,the gas-permeable membrane is in direct contact with and sealed onto thesurface of the one or more feedstock, optional liquid media, andinoculum present in the container. In some embodiments an optionalculturing media is present.

In some embodiments the gas-permeable membrane is composed of apolymeric material, such as polypropylene, polyethylene,polytetrafluorethylene, polycarbonate, polyamide, polypyrrolone,poly(amidoamine) dendrimer composite, cellulose acetate,butadiene-acrylonitrile, TeflonAF2400, and nylon. In some embodimentsthe pore size for the gas-permeable membrane ranges from 0.05-1.5 μm,such as 0.2 μm, 0.45 μm, and 1.0 μm. In some embodiments thegas-permeable membrane is in the form of a sterile cloth-like materialwhile in others the membrane is in the form of a paper-like material. Insome embodiments the surface is smooth in texture, in others the surfaceis rough in texture. In some embodiments the path for gas diffusion isessentially direct while in others the path is tortuous.

In some embodiments the reactor produces a biofilm-biomat that serves asa food source, such as a protein source and/or an oil source. In otherembodiments the biofilm-biomat serves as a leather analog and/or abioplastic. In still other embodiments the biofilm-biomat serves as asource of biofuel precursors or as a biofuel itself. In yet otherembodiments, the biofilm-biomat serves to produce organic products suchas organic acids, antibiotics, enzymes, hormones, lipids, mycotoxins,vitamins, pigments and recombinant heterologous proteins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Growth of Fusarium oxysporum strain MK7 biomat in nutrientmedium that was refreshed daily after the initial 4-day biomat formationstage.

FIG. 2. Three-centimeter-thick biomat of Fusarium oxysporum strain MK7that was formed in liquid nutrient medium that was refreshed daily(after day 4). Nylon mesh screen underneath the biomat is shown and usedfor lifting and moving the biomat to fresh medium.

FIG. 3. Continuous flow system designed to continuously feed Fusariumoxysporum strain MK7 biomat growth and remove nutrients from media.White biomats shown in channels after 7 days of growth from the time ofinoculation.

FIG. 4. Biomat growth after 10 days of growth from the time ofinoculation (6 days under continuous flow+4 days under quiescent/staticconditions).

FIG. 5. Semi-continuous production of biomat showing (A) removal of themost mature portion of the biomat at day 12. After harvesting ⅓ of themost mature biomat at the lower end of the tray, the remaining biomat isphysically moved down in the direction of the arrow until the edge ofthe biomat touches the end of the tray (B). Moving the biomat creates afresh open space at the upper end of the tray where new biomat forms.

FIG. 6. Cumulative production of biomass over time using thesemi-continuous production method. Dashed line is the linear regressionline for day 5 through day 19 (y=0.57x−1.52, r²=0.973). Error bars arestandard deviations of the mean of three replicate trays. Error bars arenot visible when smaller than data point symbol.

FIG. 7. Continuous production of biomat showing removal of the mostmature portion of the biomat at the right. While continuously harvestingthe most mature biomat at the right side of the tray, fresh open spaceis created at the left end of the tray enabling new biomat to form.Liquid medium in the tray can be replenished and/or augmented asrequired or continuously.

FIG. 8. Orange pigmentation of Fusarium oxysporum strain MK7 biomats(two excised disks at the right) after irradiation with UVB light forfour hours. Two excised disks from non-irradiated control biomats areshown at the left.

FIG. 9. Field emission scanning electron microscopy of 4 day oldFusarium oxysporum strain MK7 biomats produced using MK7-1 medium(described in PCT/US2017/020050) with glycerol, corn starch and cornsteep liquor. Images A, B and C show biomat with EPS matrix removed byethanol washing. A) View of top surface of biomat with aerial hyphae. B)Cross-section of the dense bottom layer with arrow delineating thelayer. The cross-sectional view was created by cutting the biomat with arazor blade. The bottom of the biomat is shown at the bottom left cornerof the image and the poorly adhering transition layer above the densebottom layer is shown at the upper right corner. C) View of bottomsurface of biomat. D) View of bottom surface of biomat with EPS matrixin place (i.e., EPS not removed with ethanol wash).

FIG. 10. Transmitted light microscope images (100×) of biomats grown onglycerol, starch and corn steep liquor. The image at the left of theaerial hyphal layer reveals the predominant near-vertical orientation ofthe filaments. The image at the right shows the dense bottom layer andthe adjacent transitional layer.

FIG. 11. A: Chicken breast on the left and fresh biomat with similartexture grown on glycerol on the right. B: “MycoBurger” prepared byrenown chef Brooks Headley using fungal biomat.

FIG. 12. Biomats produced using the disclosed method. A: Reishimushroom; B: Pearl Oyster mushroom; C: Blue Oyster mushroom; D:Cauliflower mushroom; E: Elm oyster mushroom; F: Giant Puffballmushroom.

FIG. 13. A. Chicken nugget produced from Fusarium oxysporum strain MK7biomat grown on a mixture of glycerol, starch and corn steep liquor. B.Chicken nugget produced from giant puffball biomat grown on malt media001 (40 g malt, 4 g peptone, 1.2 g yeast extract, 20 drops/1 ml canolaoil, 4 g ground oats, 1000 mL water).

FIG. 14. Yogurt prepared from a live yogurt culture using A. 25% MK7liquid dispersion:75% whole milk, B. 50% MK7 liquid dispersion:50% wholemilk, and C. 100% MK7 liquid dispersion. The MK7 liquid dispersion usedin these cultures was prepared from Fusarium oxysporum strain MK7biomats grown on acid whey.

FIG. 15. Vegan ice cream analogue produced from Fusarium oxysporumstrain MK7 biomats.

FIG. 16. Formation of biofilm-biomat in the encapsulated reactor startswhen cells attach to the gas-permeable membrane where oxygen is readilyavailable. Over time, biofilm-biomat grows downward and ultimately fillsthe space of the reactor, consuming all liquid and nutrients.

FIG. 17. Fusarium oxysporum strain MK7 biomats grown in five days understatic conditions in Petri dishes covered with semi-permeable membranesconstructed with (A)-(C) polypropylene and (D) polycarbonate.Essentially no free liquid remained in the Petri dish and all nutrientswere incorporated into the biomat. The void/liquid volume of the reactorwas essentially filled with biomat.

FIG. 18. An attached bag separated from the liquid medium by agas-permeable membrane is used to supply and capture gasses. Theintegrated multi-functional membrane allows for ingress of oxygen andegress of CO₂ and other produced gases. Fungal biomass grown in thelower liquid compartment (yellow) converts the feedstocks and nutrientsinto biomat that fills the compartment as it grows. The denseconsolidated biomat can be easily harvested by opening the reactorclosure system (e.g. Zip-Lock® type) and removal from the bag.

FIG. 19. Basic hermetic reactor (1). Multiple channels (4) with sharedwalls/baffles (9), front valves (6) and back valves (8) and a gaspermeable membrane (2) are shown.

FIG. 20. Basic hermetic reactor (1) with a single gas collection chamber(14).

FIG. 21. Basic hermetic reactor (1) with channeled gas collectionchambers (15) having gas specific channels (30, 40).

FIG. 22. Basic hermetic reactor (1) with channeled gas collectionchambers (15) having gas specific channels (30, 40) with gas specificpermeable membranes (2, 50).

FIG. 23. Basic hermetic reactor (1) with cylindrical channels (4),walls/baffles (9), front valves (6) and back valves (8) and a gaspermeable membrane (2).

DETAILED DESCRIPTION

Edible filamentous fungi can be used as a protein source, either aloneor incorporated into foodstuffs. For example, the protein content forpearl oyster mushrooms is 27.25%, for blue oyster mushrooms 24.65%, forreishi mushrooms 15.05% (Stamets (2005) Int J Medicinal Mushrooms7:103-110), for giant puffballs 27.3% (Agrahar-Murugkar and Subbulakshmi(2005) Food Chem 89:599-603), and for cauliflower mushrooms 32.61%(Kimura (2013) BioMed Res. Int. Article ID 982317).

Yet while the fruiting bodies of Basidiomycota filamentous fungi, suchas Agaricus bisporus (crimini and white), Boletus edulis (porcini),Cantarellus cibarius (chanterelle), Ganoderma lucidum (Reishi),Morchella species (Morel), Hypsizygus tessellatus (clamshell), Pleurotusostreatus (pearl oyster and blue oyster), Pleurotus eryngii (trumpetroyale), Pholiota microspora (forest nameko), Sparassis crispa(cauliflower), Hypsizygus ulmarius (elm oyster), Cyclocybe aegerita(velvet pioppini), Grifola frondosa (maitake), Lentinula edodes(shiitake), Laetiporus species (chicken of the woods), Calvatia gigantea(giant puffball), and Tuber species (truffles) are commonly used infoodstuffs, there are few products primarily comprising the vegetativemycelia of either the Basidiomycota or Ascomycota filamentous fungi.This is due, in part, to mycelia typically being either subterraneous orlargely inseparable from the matter on which it grows.

Yet under particular conditions, filamentous fungi can form fungalbiomats via surface fermentation under anaerobic, microaerobic, oraerobic conditions or a combination thereof. Here, the filamentousfungal biomats comprise the fungal species and/or strain and/or progenythereof primarily in the form of mycelia, fragments of mycelia, hyphae,fragments of hyphae, and to a lesser extent contain conidia,microconidia, macroconidia, or any and all combinations thereof and insome cases can also contain pycnidia and chlamydospores.

Typically, the filamentous biomats are primarily comprised of mycelia;that is, a complex network of interwoven vegetative hyphae filaments.The average length of non-broken filaments within the biomat isgenerally at least 0.1 mm, such as between 0.1 mm-0.5 mm, 0.5 mm-50 cm,0.6 mm-40 cm, 0.7 mm-30 cm, 0.8 mm-25 cm, 1.0 mm-20 cm, 1.4 mm-15 cm,1.6 mm-10 cm, 1.7 mm-8 cm, 1.8 mm-6 cm, 2.5 mm-4 cm, and 5 mm-2 cm, 2cm-25 cm, 4 cm-30 cm, 5 cm-40 cm, 6 cm-50 cm, 8 cm-60 cm, 10 cm-100 cm.

The growth of filamentous fungal biomats can be accomplished via surfacefermentation. This involves inoculating liquid media containing a carbonsource and a nitrogen source with filamentous fungal cells. Suitablecarbon sources are sugars (e.g. sucrose, maltose, glucose, fructose,Japan rare sugars, etc.), sugar alcohols (e.g. glycerol, polyol, etc.),starch (e.g. corn starch, etc.), starch derivative (e.g. maltodextrin,cyclodextrin, glucose syrup, hydrolysates and modified starch), starchhydrolysates, hydrogenated starch hydrolysates (HSH; e.g. hydrogenatedglucose syrups, maltitol syrups, sorbitol syrups, etc.), lignocellulosicpulp or feedstock (e.g. sugar beet pulp, agricultural pulp, lumber pulp,distiller dry grains, brewery waste, etc.), corn steep liquors, acidwhey, sweet whey, milk serum, wheat steep liquors, carbohydrates, foodwaste, olive oil processing waste, hydrolysate from lignocellulosicmaterials, and/or combinations thereof. The filamentous fungal cellsgenerate biomats which are located on the surface of the growth media;that is, they essentially float atop the growth media.

In many cases, especially for Ascomycota fungi, growth media wasinoculated with an inoculum comprising planktonic filamentous fungalcells. High quality inoculum is composed of planktonic cells, which aredefined as single cells that are not clumped or aggregated together, arepreferably isolated from an exponential growth phase, and can includemicroconidia. Ideally, the cells of the inoculum float on the surface ofthe growth media, such as those cells having a high lipid content, andresult in increased growth rate. Cells or clumps of cells that aresubmersed within the growth media negatively affect the cells floatingon the surface and the biomats they form. Specifically, the biomatsresulting from growth media containing a significant number of clumpedsubmersed cells are typically discolored and tend to not growhomogeneously dense mats.

For Basidiomycota spore inoculation, approximately 2 cc of sterilespores suspended in deionized water from a spore syringe (e.g.MycoDirect, Huntley, Ill.) are used to inoculate approximately 75 mL ofgrowth media in small Pyrex trays. Alternatively, 1 cc of sporessuspended in deionized water from a spore syringe was plated on acontainer having malt extract agar media+CF (30 g dry malt extract, 20 gagar, 1000 mL water+0.01% chloramphenicol) using standard sterileconditions. Containers were sealed with parafilm and incubated at roomtemperature until mycelium completely covered the surface of the agar. Asegment of mycelium from the agar preparation approximately 2 cm inwidth cut into a wedge was then diced into the smallest size possiblebefore transferring to a tube with growth media. Liquid culture tubeswere sealed, incubated at room temperature, and shaken by hand or shakenby mechanical means (i.e. continuous shaking or a continuous stirredtank reactor) for about 1 minute at least five (5) times per day tobreak up mycelium as much as possible. Liquid cultures were incubateduntil visually turbid, typically three or more days. The liquid cultureswere then used to inoculate growth medium in trays at a 10% or 15% oftotal growth medium volume.

Basidiomycota fruiting bodies were also used to generate inoculum forinitiating filamentous biomats. In some instances, inoculum was preparedby (a) surface sterilizing fruiting bodies, for example in a 5% bleachsolution, (b) rinsing with sterile media, (c) grinding under sterileconditions to either less than 5 mm long aggregates or greater than 5 mmaggregates, depending on the final use, (d) surface sterilizing theground mushroom biomass for example in a 5% bleach solution, and againrinsing with sterile media. 5 grams of the ground surface-sterilizedfruiting body biomass was used directly as inoculum. In other instances,a pure culture derived from a fruiting body was used. Here, ˜3 mm³portions of fruiting body was placed on agar media containing 0.01%chloramphenicol and incubated at room temperature. After 2-5 days ofgrowth, hyphae were transferred onto fresh agar+chloramphenicol mediaand grown for another 3-7 days. Culture purity was confirmed byextracting and purifying DNA (FastDNA Spin Kit, MP Biomedicals),sequencing the 16S rRNA sequence and/or ITS region, and performingphylogenetic classification of the sequences using Blast (NCBIdatabase). Upon confirmation, hyphae were used to inoculate 50 mL ofsterile liquid media and agitated/rotated at 185 rpm for approximately 5days before using as inoculum at a ratio of about 7.5% inoculum to 92.5%liquid media.

While a number of different media can be used, some media is not welladapted for growth of filamentous fungal biomats, such as Hansen's media(per liter=1.0 g peptone, 0.3 g KH₂PO₄.7H₂O, 2.0 g MgSO₄.7H₂O 5.0 gglucose with a C:N ratio of 26.9) upon which full, cohesive biomats werenot produced. Those media which work exceptionally well include MK7A,MK7-1, MK7-3 (all described in WO 2017/151684), as well as the mediapresented below.

Malt Medium 001 (C:N ratio of 19.1)

Ingredient Amount Grade Light Pilsner  40.0 g Food Malt Peptone   4.0 gResearch Yeast Extract   1.2 g Research Powder Canola Oil   1.0 mL FoodGround Oats   4.0 g Food Tap H₂O  1000 mL N/A

MK-7 SF Medium (C:N ratio of 7.5)

Ingredient Amount Grade NH₄NO₃ 7.553 g ACS Urea 2.548 g USP CaCl₂ 2.000g Reagent MgSO₄ * 2.000 g USP 7H₂O KH₂PO₄ 7.500 g Reagent Trace * 2.000mL * Glycerol 0.075 Kg Food/USP Yeast Exract 1.750 g Research FeCL₂ *4H₂O 0.020 g Reagent DI H₂O 0.940 L N/A

Trace Components * Micronutrients * mg/L Grade FeSO4•7 H2O 9.98 ACSZnSO4•7 H2O 4.4 USP/FCC MnC12•4 H20O 1.01 Reagent CoC12•6 H2O 0.32Reagent CuSO4•5 H2O 0.31 Technical (NH4)6Mo7O24•4 0.22 ACS H2O H3BO30.23 ACS EDTA, free acid 78.52 Electrophoresis

Malt Media 001 Supplemented with NH₄NO₃(C:N ratio of 7.5)

Ingredient Amount Grade NH₄NO₃   5.0 g ACS Light Pilsner  40.0 g FoodMalt Peptone   4.0 g Research Yeast Extract   1.2 g Research PowderCanola Oil   1.0 mL Food Ground Oats   4.0 g Food Tap H₂O  1000 mL N/A

Osmotic pressure readings were taken by sterilely removing 250 μl ofmedia and using a recently calibrated Osmometer (Model 3250 SN:17060594) capable of measuring up to 5000 mOsm. Three reading were takenand provided the following results: Hansen's=39, 39, 38; Malt 001=169,168, 169; MK-7 SF=1389, 1386, 1387; Malt 001+NH₄NO₃=288, 287, 286.

In addition, the media used in our method can define the protein contentof the resulting biomat. For example, while the natural protein contentof the fruiting body of Blue Oyster mushrooms is reported to be 24.65%(Stamets (2005) Int J Medicinal Mushrooms 7:103-110) Blue Oyster biomatsgrown according to our method on Malt 001 media have a higher moisturecorrected protein content of 29.82%, an increase in protein content of5.71%. More strikingly, the protein content of fruiting bodies of GiantPuffball is reported to be 27.3% (Agrahar-Murugkar and Subbulakshmi(2005) Food Chem 89:599-603), yet Giant Puffball biomats grown with ourmethod on Malt 001 media have a moisture corrected protein content of32.04%, while MK7-1 SF media produces a moisture corrected proteincontent of 46.33% and Malt 001+NH₄NO₃ media produces a moisturecorrected protein content of 46.88%, essentially an increase in proteincontent of 19.85% over that reported by Agrahar-Murugkar andSubbulakshmi.

Harvesting of biomats typically occurs after 2-3 days of growth,although in some instances longer growth periods are desirable, such aswhen thicker or denser biomats are desired/required. For example, growthperiods of 3.5-4 days, 3-5 days, 4-6 days, 5-7 days, 6-9 days, 7-10days, 19-21 days, or even up to 2 months may be desirable. Due to thecohesive structure of the filamentous biomats grown under surfacefermentation conditions described in PCT/US2017/020050 and herein, thefilamentous biomats have enough tensile strength to be liftedessentially intact from the surface of the media at the end of thegrowth period. Table 1 presents some examples tensile strength measured.

TABLE 1 Average Tensile Strength for some filamentous fungal biomatsAvg. Avg. Break Tensile Thickness Width wt Strength Organism C source(cm) (cm) (g) (g/cm²) Giant Puffball Malt 0.13 1.2 47.12 314.13 Glycerol0.10-1.3 1.2 29.05 214.85 MK7-1SF 0.25-0.35 0.65-0.8 30.67 263.98 Malt +0.09-0.10 0.9-1.1 27 281.15 NH₄NO₃ Cauliflower Malt 0.15-2.0 1.0-1.2101.05 507.38 Glycerol 0.09-0.20 1.2 202.17 242.91 Reishi Malt 0.51.0-1.2 101.05 1854.54 Blue Oyster Malt 0.5 1.2 43.40 72.74 Glycerol 0.41.3 19.04 37.27 Pearl Oyster Malt 0.5 1.0-1.2 56.7 98.96 Elm Oyster Malt0.35 1.2 50.28 143.67 F. oxysporum Glycerol 0.5-0.8 1.0 >742 >570 strainMK7

Surface fermentation can be carried out under various conditions,including static media conditions (as described in PCT/US2017/020050),semi-static media conditions, and continuous media flow conditions.

Growth under semi-static media conditions means that at least a portionof the medium is replaced before the filamentous fungal biomat isharvested. These conditions allow linear dry biomass production from day4 through day 18 (r²=0.995), after which biomass weight stabilizes atabout 2.5 Kg dry/m².

Biomats can also be produced under continuous media flow conditionswhere biomat growth is confined to the surface of the growth media wherethe medium underneath the mat is continuously refreshed orsemi-continuously refreshed.

In some instances, however, it is desirable to harvest the growingbiomat on a semi-continuous basis. Here, removal of some portion of thebiomat occurs and the remaining portion is then physically moved to theopen area of medium that was created by removal of the portion ofbiomat. This can be accomplished by physically grasping the biomat andpulling it until it touches the end of the surface fermentationcontainer or by other mechanical means. The resulting open area is thenavailable for new biomat growth without a separate or additionalinoculation step since the medium already contains viable fungal cells.This process can be repeated periodically, which can be particularlyuseful when the medium is refreshed or nutrients that have becomelimited are reintroduced.

Biomat harvesting can also be done on a continuous basis. Continuousremoval can be facilitated by a number of mechanisms. One such exampleis a roller wheel that is attached to the mature end of the biomat (seeFIG. 7). The roller wheel slowly turns and harvests the mature biomatand at the same time creates open medium for growth of new biomat at theother end of the surface fermentation container. A typical rate ofharvesting is 1.56 cm/day, although this can be altered for particularneeds or as desired by a user.

Growth under membrane encapsulated/hermetically sealed bioreactorconditions involves encapsulating liquid growth medium with no gasheadspace in an appropriate system/container. Appropriatesystems/containers are, for example, trays, Petri dishes, or anycontainer having a relatively large surface area to depth ratio. Gaspermeable membranes are placed directly on the surface of the liquidmedium and sealed tightly to the system/container. Appropriate membranesinclude, for example, polypropylene membranes (e.g. KC100 Kimguard,Kimberly-Clark, Roswell, Ga.), polyester membranes, polycarbonatemembranes, silicone membranes, polyamide membranes, cellulose membranes,and ceramic membranes, to name but a few. Gas exchange between thegrowing biomats and the surrounding atmosphere occurs solely through thesemi-permeable membrane.

In some cases, UVB light (290-320 nm) can trigger pigment production byfilamentous fungi, such as for Fusarium oxysporum strain MK7, producinga pigmented biomat. In addition to a color change, which can be usefulfor creating various food effects, treatment with UVB convertsergosterol present in the fungal cell membranes into vitamin D2 andincreases production of carotenoids, such as beta carotene andastaxanthin. Consequently, irradiating filamentous fungi, such asFusarium oxysporum strain MK7, with UVB can be used to increase vitaminD2 and carotenoids in the resulting biomats.

In some cases, the filamentous fungal biomats formed are composed oflayers of cells which are uniform in appearance, one surface of thefilamentous biomat in contact with the air and one surface in contactwith the synthetic media. In other cases, at least two distinct layersare present: an aerial hyphae layer at the top surface and a densemulticellular bottom layer in contact with the synthetic media.Oftentimes three distinct layers are present: (a) an aerial hyphae layerat the top surface, (b) a dense bottom layer and (c) a transitionallayer between the top and bottom layers. The transitional layer may beonly loosely attached to the dense bottom layer, in those cases enablingeasy separation of the bottom layer from the rest of the biomat.Filament densities of the transitional layer range from slightly lessdense than the bottom layer in the zone where the two layers meet, to adensity that is comparable to the aerial hyphae near the top of thebiomat.

Inactivation of Filamentous Fungal Biomats

The inactivation process begins with biomats harvested at least 2 daysafter cultivation. While biomats can be rinsed to remove excess growthmedia, biomat rinsing is not required, although in some cases theremoval of growth media or excess growth media is preferable. Similarly,biomats can be squeezed to remove excess growth media, again notrequired, but which may be preferable for some applications.

Elimination of cell viability and the potential of further biomat growthis desired in some instances, such as for use of the biomat as astand-alone protein source or a protein ingredient in foodstuffs. Thiscan be accomplished by heating, irradiation, and/or steaming.

For the heating process, filamentous fungal biomats can be treatedaccording to WO 95/23843 or British Patent No 1,440,642, for example, orincubated at temperatures that destroy the vast majority of theorganism's RNA without adversely affecting the organism's proteincomposition.

In irradiation, filamentous fungal biomats are exposed to ionizingenergy, such as that produced by ⁶⁰Co (or infrequently by ¹³⁷Cs)radioisotopes, X-rays generated by machines operated below a nominalenergy of 5 MeV, and accelerated electrons generated by machinesoperated below a nominal energy of 10 MeV.

Steaming is the preferred method for inactivating some filamentousfungal biomats, such as those produced by Fusarium oxysporum strain MK7and F. venentatum, as steaming can also remove some specific metabolitesfrom the biomat construct if those metabolites are produced. Here,biomats are placed such that biomat excreted liquids and condensed steamcan easily drip away from the biomats. Suitable biomat holding systemsinclude porous plastic mesh and porous trays. Other biomat holdingsystems include, but are not limited to, systems that secure the biomatin a vertical position, such as systems with a clamping mechanism thatclamps at least one end of a biomat while the remaining end(s) of thebiomat hang from said clamp and mesh systems which clamp at least twosides of the biomat, to name but a few.

Biomats are positioned within a steamer such that heated steam, such assteam of a temperature greater than 85° C., for example 95° C., comesinto contact with the biomats. In those cases where multiple trays areplaced in a single steamer, for example one tray above the other, it ispreferred to protect a lower positioned biomat from the drippings of ahigher positioned biomat. Protection should be of a form which allowssteam to contact biomats, thereby de-activating biomat viability, and toalso deflect biomat excreted liquids and condensed steam produced at ahigher level in the steamer from contacting biomats positioned at alower level in the steamer. In one embodiment, a cone is positionedbetween an upper tray and a lower tray to accomplish this result. Inother embodiments, separation between upper and lower trays also includeat least one other geometric shape such as a cylinder, a cube and/orcuboid, a pyramid, a sphere, a tori, and/or other platonic solids. Inyet another embodiment, trays are separated using at least one cylinder,cube and/or cuboid, pyramid, sphere, tori, other platonic solid, orcombinations thereof.

Biomats are steamed at least to the point where biomat viability isreduced such that further biomat growth and/or cellular reproductionwithin a biomat is negligible. Biomat viability is a function of theoriginal substrate, biomat development, steam/heat transfercharacteristics, biomat position in a steamer and biomat orientationrelative to evolved steam. As an example, Fusarium oxysporum strain MK7biomats grown on a glycerol or acid whey substrate are non-viable after5 minutes, and in some cases less than 5 minutes, of steaming. Steamedmats can be rinsed and/or squeezed to remove mat excretions andcondensed steam.

The inactivated edible filamentous fungal biomats can be used directlyas a protein source, for example in preparing foodstuffs largelycomparable to tofu, bacon, and jerky, to name but a few.

The inactivated edible filamentous fungal biomats can also be sizereduced for use as a protein source in foodstuffs. The size reductioncan occur by mechanical means such as cutting, chopping, dicing,mincing, grinding, blending, etc. or via sonication and is conductedprior to mixing with other ingredients or liquids. Size reducedparticles can be uniform in size or variable. Typically, the length ofthe sized reduced particles is between 0.05-500 mm, the width is between0.03-7 mm, and height is between 0.03-1.0 mm. For example, flour-typeparticles typically range between 0.03 mm and 0.4 mm, jerky-typeparticles range between 100 mm and 500, etc. Larger size particles canbe produced, biomats have been grown in inflatable pools (66″ indiameter) producing a single biomat 66″ in diameter and completelyround. Larger vessels can be used to grow even larger mats.

The number of size reduced particles produced per biomat is dependent onthe initial biomat size and the purpose for which the biomat sizereduced particles will be used.

Depending on the foodstuff, the size reduced particles contain averageunbroken filament lengths of at least 0.1 mm, such as between 0.1 mm-2.0mm, 0.5 mm-10 cm, 0.5 mm-30 cm, 0.8 mm-25 cm, 1.0 mm-20 cm, 1.4 mm-15cm, 1.6 mm-10 cm, 1.7 mm-8 cm, 1.8 mm-6 cm, 2.5 mm-4 cm, 5 mm-2 cm,0.5-2.5 mm, 0.5-1.8 mm, 0.5-1.7 mm, 0.5-1.6 mm, 0.5-1.4 mm, 0.5-1.0 mm,0.5-0.8 mm, 0.5-0.6 mm, 0.6-2.5 mm, 0.6-1.8 mm, 0.6-1.7 mm, 0.6-1.6 mm,0.6-1.4 mm, 0.6-1.0 mm, 0.6-0.8 mm, 0.8-2.5 mm, 0.8-1.8 mm, 0.8-1.7 mm,0.8-1.6 mm, 0.8-1.4 mm, 0.8-1.0 mm, 1.0-2.5 mm, 1.0-1.8 mm, 1.0-1.7 mm,1.0-1.6 mm, 1.0-1.4 mm, 1.4-2.5 mm, 1.4-1.8 mm, 1.4-1.7 mm, 1.4-1.6 mm,1.6-2.5 mm, 1.6-1.8 mm, 1.6-1.7 mm, 1.7-2.5 mm, 1.7-1.8 mm, or 1.8-2.5mm, as well as larger size distributions such as between 0.1-1.0 cm,0.5-2.0 cm, 1.0-5.0 cm, 2.0-7.0 cm, 5.0-10.0 cm, 7.0-20 cm, 10.0-50.0cm, and 15.0-100.0 cm.

Size reduced particles of the filamentous fungal biomats also containbroken filaments and, in some cases, broken filaments are primarilypresent, such as 100% broken filaments, 99% broken filaments, 98% brokenfilaments, 97% broken filaments, 96% broken filaments, and 95% brokenfilaments. Again, the size of the broken filaments is selected for theultimate foodstuff produced. Average broken filament lengths can rangefrom at least 0.01 mm, such as between 0.01-0.10 mm, 0.05-0.20 mm,0.1-1.0 mm, 0.50-2.5 mm, 1.0-5.0 mm, 2.0-10.0 mm, 5.0 mm-15.0 mm, 10.0mm-1.0 cm, 1.0 cm-5.0 cm, 5.0 cm-10.0 cm, 0.3 mm-30 cm, 0.8 mm-25 cm,1.0 mm-20 cm, 1.4 mm-15 cm, 1.6 mm-10 cm, 1.7 mm-8 cm, 1.8 mm-6 cm, 2.5mm-4 cm, 5 mm-2 cm, 0.3-2.0 mm, 0.3-1.8 mm, 0.3-1.7 mm, 0.3-1.6 mm,0.3-1.4 mm, 0.3-1.0 mm, 0.3-0.8 mm, 0.3-0.6 mm, 0.3-0.5 mm, 0.6-2.5 mm,0.6-1.8 mm, 0.6-1.7 mm, 0.6-1.6 mm, 0.6-1.4 mm, 0.6-1.0 mm, 0.6-0.8 mm,0.8-2.5 mm, 0.8-1.8 mm, 0.8-1.7 mm, 0.8-1.6 mm, 0.8-1.4 mm, 0.8-1.0 mm,1.0-2.5 mm, 1.0-1.8 mm, 1.0-1.7 mm, 1.0-1.6 mm, 1.0-1.4 mm, 1.4-2.5 mm,1.4-1.8 mm, 1.4-1.7 mm, 1.4-1.6 mm, 1.6-2.5 mm, 1.6-1.8 mm, 1.6-1.7 mm,1.7-2.5 mm, 1.7-1.8 mm, or 1.8-2.5 mm.

In some cases, the average broken filament length in the reducedparticles of the filamentous fungal biomats is less than 1 um, such asless than 950 nm, less than 900 nm, less than 850 nm, less than 800 nm,less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm,less than 550 nm, less than 500 nm, or less than 400 nm.

The reduced particle size of the filamentous fungal biomat can be addedas a protein source to augment the protein content of a foodstuff or canbe the sole protein component. For foods composed entirely offilamentous fungal biomats, the size reduced particles can be optimizedfor particular textures, mouth feel, and chewiness. For example, afilamentous fungal biomat food shaped and seasoned to resemble ahamburger can have 90% of the particles with lengths less than 1.5 mmand the majority of lengths being 1 mm or less, widths of less than 1mm, and depths of less than 0.75 mm. This type of food is characterizedas having a higher perceived density in the mouth, is easier to chew,offers a creamy mouth feel and a more refined food experience. Highlyprocessed biomat particles have been compared to the type of burgerfound in fine dining establishments. For a more heartier food experiencesimilar to the type of burger prepared commonly found in burgerrestaurants or BBQ's, 90% of the particles have lengths between 4 mm and10 mm, widths of 1.0 mm to 3 mm, and depths of less than 0.75 mm. Theability to alter texture, mouth feel, and chewiness allow customizationto accommodate individuals having particular dietary needs, such asthose that have trouble chewing, or who require/desire softer foodswhile still providing the same nutritional and taste experience or thosewho desired food with more texture, more mouthfeel and more mastication.Because of the ability to easily control the particle size, foodsaugmented with filamentous fungal biomats or made solely fromfilamentous fungal biomats have textures very similar to the standardprotein foods that they emulate, as can be seen in Table 2.

TABLE 2 Results from Stable Micro Systems TA XT plus texture analyzerAvg. Max Avg. Area Avg. Mean Food Hardness (g/mm) (g) Parameters FishStick Pre-Test Speed: 2.00 mm/sec Commercial 3654 ± 1774 17868 ± 5674 894 ± 284 Test Speed: 4.00 mm/sec fish stick Post-Test Speed: 10.00mm/sec MK7 fish stick 1618 ± 180 19990 ± 610 1000 ± 100 Target Mode:Distance Chicken Force: 100.0 g Nugget Distance: 20.000 mm Commercial3838 ± 56.8 27329 ± 3663 1367 ± 183 Strain: 10.0% chicken nugget TriggerType: Auto (Force) Quorn chicken 4013 ± 1066.3 27751 ± 1346.4 1415 ±111.4 Tigger Force: 5.0 g nugget Probe: HDP/WBV MK7 small 3127 ± 19.733065 ± 3458 1654 ± 173 Warner Bratzler V Slot Blade particle MK7 medium2514 ± 663 27217 ± 6437 1361 ± 322 particle MK7 large 3461 ± 77.8 34591± 2971.2 1730 ± 14.6 particle Burger 100% Beef 4326 ± 714 12350± 1727 ±14.1 burger 46.1 90% Beef, 10% 5011 14048 1929 MK7 80% Beef, 20% 2615±199 10641±511 1456 ±46 MK7 70% Beef, 30% 2240 ±262 9859 ±2947 1291 ±300MK7 60% Beef, 40% 2094± 156 8118±1088 1155±180 MK7 100% MK7, 2228 ± 5079±964 1089 ±70.6 chopped (highly 1988 processed) Firmness Food (g)Chocolate Pre-Test Speed: 1.00 mm/sec Mousse Test Speed: 1.00 mm/secNestle 182.45 Post-Test Speed: 10.00 mm/sec chocolate Target Mode:Distance mousse T.A. Variable No: 5: 0.0 g MK7 chocolate 135.09Distance: 10.000 mm mousse Strain: 10.0% Trigger Type: Auto (Force)Tigger Force: 5.0 g Probe: P/25; 25 mm DIA Cylinder Aluminum

Examples of foods that can be produced using only the reduced particlesize of the filamentous fungal biomat, with or without added flavorings,and/or that can be augmented with the reduced particle size of thebiomat are meat products (such as ground beef, ground chicken, groundturkey, chicken nuggets, fish sticks or patties, jerky, snacks (e.g.chips), soups, smoothies, beverages, milk analogues, breads, pastas,noodles, dumplings, pastries (e.g. Pate a Choux), cookies, cakes, pies,desserts, frozen desserts, ice cream analogues, yogurt, confections, andcandy.

Foods augmented with the reduced particle size of the filamentous fungalbiomat can significantly increase the protein content, which isparticularly important for individuals following a vegan diet. Forexample, augmenting a cup of soup (227 g) with 68.1 g of MK7 liquiddispersion (i.e. 1 part MK7 to 3 parts water) adds 8.5 g of protein andaugmenting a bowl of soup (340 g) with 136 g of MK7 liquid dispersionadds 17 g of protein. Use of MK7 liquid dispersion as the primaryingredient, such as in vegan soups, drinks, smoothies, etc. furtherincreases the protein content of these foods. Changing the MK7 to waterratio will in turn change the degree of protein augmentation.

Whether the reduced particle size of the biomat is used to augment theprotein content of food or is used as the sole protein component, insome instances binders are helpful in achieving the desired texture.Approved foodstuff binders are envisaged, such as egg albumen, gluten,chickpea flour, vegetarian binders, arrowroot, gelatin, pectin, guargum, carrageenan, xanthan gum, whey, chick pea water, ground flax seeds,egg replacer, flour, Chia seeds, psyllium, etc which can be usedsingularly or in combination. In addition to foodstuff binders, thereduced particle size of the filamentous fungal biomat can also be mixedwith approved flavors, spices, flavor enhancers, fats, fat replacers,preservatives, sweeteners, color additives, nutrients, emulsifiers,stabilizers, thickeners, pH control agents, acidulants, leaveningagents, anti-caking agents, humectants, yeast nutrients, doughstrengtheners, dough conditioners, firming agents, enzyme preparations,gasses, and combinations thereof. Typically, binders, flavors, spices,etc. are selected to meet the demands of a particular population. Forexample, milk and/or milk solids are not used to accommodate individualswith dairy allergies/sensitivities, wheat flour may not be used toaccommodate those with gluten allergies/sensitivities, etc.

In some applications, the reduced particle size filamentous fungalbiomat is used in foodstuffs that simulate, chicken nuggets, turkey,pork, fish, burgers, sausages, jerky, bacon, and the like. Here, asingle type of reduced particle size filamentous fungal biomat can beused or a variety of reduced particle sizes. Similarly, the reducedparticle sizes can be from a single source of filamentous fungal biomator from a combination of different sources of filamentous fungalbiomats; e.g. MK7 alone or MK7+Giant Puffball biomats.

In some applications, the reduced particle size filamentous fungalbiomat is dried, ground to a sufficiently small particle size and usedas a flour for production of augmented protein baked goods, such asbread, rolls, muffins, cakes, cookies, pies, etc.

One aspect of introducing protein into a foodstuff is to use a liquiddispersion, made from the filamentous fungal biomat as a replacementingredient for milk or a milk analogue. The liquid dispersion can beused in a number of recipes including soups, ice cream, yogurt,smoothies, fudge, and candies such as caramel and truffles. In somecases, the filamentous fungal biomats produced from differentfeedstocks/carbon sources result in liquid dispersions having differentflavors. For example, when the feedstock/carbon source is glycerol, theresulting liquid dispersion produced from Fusarium oxysporum strain MK7is sweeter while a liquid dispersion resulting from Fusarium oxysporumstrain MK7 grown on an acid whey feedstock/carbon source tends to besourer. The native sweetness or sourness of the filamentous fungus, e.g.Fusarium oxysporum strain MK7, transfers to the ultimate food product.For instance, acid whey liquid dispersions lends itself to yogurt, whileglycerol liquid dispersions tends to lend itself to mouse, caramel orfudge.

The filamentous fungal biomat:water ratio can be adjusted to produce aliquid dispersion of the appropriate consistency and density. Ratios canbe from 1:2 to 10:1, with preferred ratios as 1:3, 1:4, and 7:3. Forexample, a relative density ratio of 1:3 is amenable to ice creamanalogues, beverages and yogurt.

In some cases, the filamentous fungal biomat can be used as a source ofoil, for example truffle oil produced from surface fermentation ediblefungal biomats of Tuber species.

The use of filamentous fungi as valuable microbial factories has beenexploited in the past, but has generally required significantinfrastructure and/or equipment, energy requirements, expensivereagents, and/or significant human resources. Filamentous fungi are wellknown for having the greatest metabolic diversity of all microorganismson Earth, including the ability to produce a wide spectrum of organicadds, antibiotics, enzymes, hormones, lipids, mycotoxins, vitamins,organic acids, pigments, and recombinant heterologous proteins (Wiebi(2002) Myco-protein from Fusarium venenatum: a well-established productfor human consumption. Appl Microbiol Biotechnol 58, 421-427; El-Enshasy(2007) Chapter 9—Filamentous Fungal Cultures—Process Characteristics,Products, and Applications. In. Bioprocessing for Value-Added Productsfrom Renewable Resources. Editor: Shang-Tian Yang. Elsevier; Gibbs et al(2000) Growth of filamentous fungi in submerged culture: problems andpossible solutions. Crit. Rev. Biotechnol. 20, 17-48), as well as theability to degrade many types of recalcitrant materials such aslignocellulose and humic substances in soils.

While widely used, significant challenges to production by submergedfermentation still exist and include important factors such as growthlimitation due to the restricted oxygen availability and excessive shearforces generated by agitation (Gibbs et al (2000) Growth of filamentousfungi in submerged culture: problems and possible solutions. Crit. Rev.Biotechnol. 20, 17-48). Since oxygen solubility in water under Earthsurface conditions is about 8 mg/L, it is readily depleted during rapidgrowth in submerged cultures. Thus, continuous aeration using complex,expensive and energy intensive aeration and agitation systems isrequired to maintain high growth rates. The cultivation of filamentousfungi is even more challenging since the filamentous morphology impartsnon-Newtonian rheological behavior that further inhibits oxygen transferto solution (Nørregaard et al. (2014) Filamentous Fungi Fermentation. InIndustrial Scale Suspension Culture of Living Cells, H.-P. Meyer, and D.R. Schmidhalter, eds. (Wiley-VCH Verlag GmbH & Co. KGaA), pp. 130-162).As culture densities increase, the amount of energy required to aerateand mix the cultures increases nonlinearly as well as the energyrequirements to aerate dense cultures are very high. For manyfilamentous species, vigorous agitation and aeration of the culturesbecomes detrimental to hyphal growth and as a result dramaticallydecreases growth rate. These and other challenges to submergedfermentation of filamentous microorganisms require innovative solutionsto effectively harness these organisms with the limited resourcesavailable in spacecraft and at extraterrestrial stations.

The disclosed hermetic reactor system (1) addresses these problems andhas the following advantages:

-   -   Active aeration or agitation of the liquid culture is not        necessary    -   In-situ aggregation of biomass into a single coherent mat with        significant tensile strength (>0.1 kg/cm of biomat width) allows        easy harvesting    -   Textured biomats can be used for a wide variety of mission        critical products (i.e. food, bioplastics, biofuels, nutritional        supplements, and as an expression platform for a variety of        pharmaceuticals    -   Minimal water use as well as minimal and/or no residual waste        water or nutrients from the process while maintaining high        biomass production (80-120 g/m²/d or 0.55 g/L/h)    -   Growth rates can translate to the production of fully formed        biomats in as little as 2 days or can be further expanded for        more than 10 days    -   High biomass density (biomats are typically 100-180 g/L)    -   A variety of filamentous fungi (including extremophiles) with        specific advantages for different processes can be grown    -   Scale-up or down is relatively straightforward and does not        result in decreased productivity.    -   Process can use a very wide variety of C and N-rich waste        substrates that arise from natural disasters and/or space        missions.

The disclosed hermetic reactor system (1) provides a self-containedbiofilm-biomat reactor comprising a container and placed within thecontainer a feedstock, a fungal inoculum, a gas-permeable membrane (2),and optionally a liquid nutrient medium. Depending upon thecircumstances, the reactor can be a one-time use reactor or a reusablereactor.

Typically, the container is capable of being sealed and may include acontainer cover in addition to a seal. Some container examples are acovered tray, a covered petrie dish, another type of covered container,or a bag. For some uses or in some environments the container has aplurality of growth chambers, for example following a manifold designand/or a baffling system. To maximize efficiency in some environmentalconditions, the container is produced from one or more feedstocks; thesemay or may not be identical to the feedstock placed within thecontainer.

The feedstock is inoculated with a fungal strain, such as an ascomycetesor basidiomycetes fungal strain. Examples of ascomycetes strains areFusarium oxysporum strain MK7 (ATCC PTA-10698 deposited with theAmerican Type Culture Collection, 1081 University Boulevard, Manassas,Va., USA), Fusarium venenatum, Fusarium avenaceum, and/or combinationsthereof. Inoculation of the feedstock can occur at the time thefeedstock is placed within the container or can occur sometime after thefeedstock has been placed. That is, the hermetic reactor (1) can beprimed with freeze-dried filamentous fungal inoculum that is revivedupon contact with the feedstock or the feedstock can be directlyinoculated after placement in the hermetic reactor channel(s) (4) or thefeedstock can be inoculated and then placed in the hermetic reactorchannel(s).

With respect to the feedstock used in the reactor, the feedstock can bea waste product, such as naturally occurring urine and/or feces, foodwaste, plant material, industrial waste such as glycerol, and wasteby-products, starch and/or by products of starch hydrolysis, acid whey,sugar alcohol, and/or combinations thereof. Synthesized or manufacturedwaste surrogates, such as surrogate human urine can also be used. Plantmaterial feedstocks are typically lignocellulosic. Some examples oflignocellulosic feedstock are agricultural crop residues (e.g. wheatstraw, barley straw, rice straw, small grain straw, corn stover, cornfibers (e.g. corn fiber gum (CFG), distillers dried grains (DDG), corngluten mean (CGM), switch grass, sugar beet pulp, waste streams frompalm oil production, hay-alfalfa, sugarcane bagasse, non-agriculturalbiomass (e.g. algal biomass, cyanobacterial biomass, urban treeresidue), forest products and industry residues (e.g., softwoodfirst/secondary mill residue, hard softwood first/secondary millresidue, recycled pater pulp sludge), lignocellulosic containing waste(e.g. newsprint, waste paper, brewing grains, used rubber tire (URT),municipal organic waste and by-products, yard waste and by-products,clinical organic waste and by-products, and waste and by-productsgenerated during the production of biofuels (e.g. processed algalbiomass, glycerol), and combinations thereof.

A gas-permeable membrane(s) (2) allows optimization of the system inseveral different ways that are illustrated in FIGS. 19-22. While thehermetic reactor system illustrated in the Figures has a total of ninechannels (4), the skilled artisan appreciates that any number ofchannels (4) can be present, from a single channel (4) to a plethora ofchannels (4), depending on the space available for placement thehermetic reactor (1). Similarly, the shape of the channels (4) is notlimited to a rectangular prisms or cylinders and can take any shapesuitable to fit the available for the hermetic reactor (1).

In some cases, the membrane (2) is placed in direct contact with thesurface of the feedstock, optional liquid media, and inoculum present inthe container as shown in FIG. 16. The membrane can also be sealed incontact with the surface of the feedstock, for example, by attaching itto a plastic frame with an integrated rubber gasket.

In other instances, the membrane is suspended over the feedstock so thatas the fungi grows and consumes oxygen, the membrane drops down towardsthe mat or onto a baffle system located between the membrane and thefeedstock which allow for growth of aerial hyphae. Such as system isshown in FIG. 19. Here, the hermetic reactor (1) is comprised ofmultiple channels (4) which initiate at an inlet valve (6) at the front(7) of the reactor, terminate at an outlet valve (8) at the back (5) ofthe reactor, and are separated by baffles/walls (9). A gas permeablemembrane (2) forms the top of the reactor. The bottom (3) of the reactorcan be formed of any suitable substance including, but not limited toboth hard and soft plastics such as polyethylene terephthalate, highdensity polyethylene, polyvinyl chloride, polyactic acid, polycarbonate,acrylic, acetal, nylon, acrylonitrile butadiene styrene, glass, metalssuch as aluminum, titanium, stainless steel etc. and/or combinationsthereof. The baffles/walls (9) can be made of similar materials.Suitable front (6) and back (8) valves include, but are not limited to,one-way valves, 2-way valves, ball valves, butterfly valves, gatevalves, plug valves, globe valves, pinch valves, disc check valves,attached valves, detached valves, and/or combinations thereof. The inletvalve (6) serves to provide access to the chamber (4) for delivery offeedstock/media to the chamber while the outlet valve (8) allows removalof exhausted feedstock and/or filamentous fungal biomat. Thegas-permeable membrane (2) can be composed of a polymeric material, suchas polypropylene, polyethylene, polytetrafluorethylene, polycarbonate,polyamide, polypyrrolones, poly(amidoamine) dendrimer composite,cellulose acetate, butadiene-acrylonitrile, TeflonAF2400, and nylon.While the pore size of the gas-permeable membrane (2) typically rangesfrom 0.05-1.5 μm, such as 0.2 μm, 0.45 μm, and 1.0 μm, the membrane (2)can be in the form of a sterile cloth-like material or the form of apaper-like material. For some uses, the membrane's surface is smooth intexture, for others the surface is rough in texture. In addition, thepath for gas diffusion can vary from being essentially direct tofollowing a more tortuous path.

In other situations, the membrane facilitates ingress of oxygen andegress of other gases produced during fungal growth (FIG. 18). In thissituation the hermetic reactor (1) has a gas collection chamber (14)that is immediately atop of the gas permeable membrane (2) (see FIG.20). The gas collection chamber (14) can be made of similar materials tothose used for the walls/baffles (9) or the bottom (3) of the reactor;i.e. both hard and soft plastics such as polyethylene terephthalate,high density polyethylene, polyvinyl chloride, polyactic acid,polycarbonate, acrylic, acetal, nylon, acrylonitrile butadiene styrene,glass, metals such as aluminum, titanium, stainless steel etc. and/orcombinations thereof. Alternatively, the gas collection chamber iscomprised of channels (15) which can mirror the channels (4) of thehermetic reactor (1) or which encompass more than one of the hermeticreactor channels (4) (see FIG. 21).

In yet other systems, separate gas permeable membranes are used foringress and egress of gases. FIG. 22 illustrates such a system. In thisinstance, two different gas permeable membranes (2, 50) feed intoseparate gas collection channels (30, 40) and are present over a singlereactor channel (4). This type of system allows ingress, egress, and/orcollection and/or separation of distinct useful gases. As an example,one membrane might be calibrated for oxygen passage and the secondmembrane calibrated for carbon dioxide or hydrogen passage or otherrelevant gas systems.

The reactor (1) produces a biofilm-biomat that serves as a food source,such as a protein source and/or an oil source. However, thebiofilm-biomat can also serve as a leather analog, a bioplastic, asource of biofuel precursors, a biofuel, and/or combinations thereof. Inyet other embodiments, the biofilm-biomat serves to produce organicproducts such as organic acids, antibiotics, enzymes, hormones, lipids,mycotoxins, vitamins, pigments and recombinant heterologous proteins.

The disclosed biofilm-biomat reactor fermentation technology enablesgrowth on standard as well as extreme feedstocks and media, such ashuman waste (urine/feces), and produces a highly consolidated andtextured product without the requirement of a separation orconcentration step. Relatively high biomass production rates (0.55 g/L/hdry biomass) and high culture densities (100-180 g/L) are achievedwithout the need for active aeration or agitation. Scale-up of thesystem vertically, horizontally, and/or in more than two dimensions issimple and does not result in decreased productivity. The producedbiofilm-biomats are typically 0.2 to 2.5 cm thick with a dry mattercontent of 10-30% and can be readily used for mission critical needssuch as meat alternatives, a myriad of other appetizing foods, andbuilding materials.

The fungal biofilm-biomats grown in the disclosed reactor system aredescribed as pellicles, which in many ways are similar to the microbialbiofilms that grow on surfaces, but are suspended in liquid culture atthe gas-liquid interface. For example, bacterial cells within biofilmshave been shown to withstand extreme disinfection treatments with sodiumhypochlorite (bleach) and sodium hydroxide (Corcoran, 2013). Thedisclosed reactor system takes advantage of the biofilm structure,enabling growth on harsh human and industrial wastes and by-productsthat may be generated under extreme conditions such as those generatedon space missions or by other harsh conditions caused by naturaldisasters.

The disclosed reactor design incorporates a gas-permeable membrane thatsits directly on or suspended just above the liquid surface. Theencapsulated reactor design allows for gas exchange with the exterioratmosphere but is hermetically sealed to keep contaminants from enteringor gases/liquids from escaping. The encapsulated reactor design can alsoenable separation of consumable gases from evolved gases by way of gaspermeable membrane. To accomplish this, in some instances valves and/oradditional porous membranes having the same or different properties areused to form distinct layers between various aspects of the one or morefeedstocks and optional liquid culture media.

Rapid biofilm-biomat growth using the disclosed reactor design has beendemonstrated with a variety of gas-permeable membrane materials. FIG. 17shows an approximately 7 mm thick biomat grown in reactor where thecontainer was a Petri dish covered with a polypropylene membrane whichwas laid directly on the feedstock/liquid medium surface. The initialbiofilm formed by direct attachment to the membrane and grew downwardinto the liquid medium over time (see FIG. 16). By the end of a five-daygrowth period, essentially all of the feedstock/liquid medium wasconsumed and dense biomass completely filled the volume underneath themembrane.

The biomat produced only mildly adheres to the membrane and was easilyharvested by simply peeling away the biomat from the membrane (see FIG.17A-D). Additional experiments with polycarbonate membranes haveproduced similar results (data not shown). Thus, the total reactorvolume can be efficiently utilized to produce dense, easily harvestedbiomass.

The biofilm-biomats commonly produced in the disclosed reactors areconsolidated (110-180 g/L) and, depending on the fungus and growthconditions, exhibit a fibrous texture. Production of a fibrous biomasscan be crucial for certain mission critical products such as foods thatrequire texture to simulate meat, as well as fibrous materials thatsimulate leather and wood. The consolidated nature of the biomass alsoenables easy harvesting without the need for a concentration step (e.g.,centrifugation, filtration).

Use of the Biofilm-Biomat Reactors in Zero Gravity

The primary physical force controlling formation and growth of thebiofilm-biomat in the disclosed reactor is attachment to the membrane.Without being bound by theory, it is believed that grown in thedisclosed reactor will not be impacted by the zero-gravity conditionsexperienced during space flight. Gravity driven directional growth orgrowth controlled by physical mixing or flow is not the overridingfactor in the system, as it tends to be in gravity environments.Previous experiments in space successfully demonstrated fungal growthEuropean Space Agency, Expeditions 25-28, Growth and Survival of ColoredFungi in Space (CFS-A)), providing an additional measure of confidencethat the disclosed reactor system will function in a space environment.

For space missions and ease of deployment, freeze dried inoculum andessential ingredients to support growth on specific feedstocks (ifneeded) can be preloaded in the reactor.

Astronauts and space travelers can then prepare the feedstock, inoculum,and any media components. Incubation time is dependent on thefeedstocks, the strain of microorganism, and other growth parameterssuch as pH, temperature and water content. The incubation conditions aresimple in that fermentation is conducted under static conditions wherethe reactor is simply allowed to incubate in place. Dense consolidatedbiomats are harvested by simply opening the reactor closure (e.g. aZiplock®-type) and removing the mats.

EXAMPLES Example 1: Growth of Strain Fusarium oxysporum Strain MK7 andOther Fungi in Static Tray Reactors

Filamentous acidophilic Fusarium oxysporum strain MK 7, Ganodermalucidum (Reishi; FIG. 1A), Pleurotus ostreatus (pearl oyster, FIG. 1B:and blue oyster, FIG. 1C), Sparassis crispa (cauliflower; FIG. 1D),Hypsizygus ulmarius (elm oyster; FIG. 1E), Calvatia gigantea (giantpuffball; FIG. 1F), and Fusarium venenatum biomats were grown in shallowstatic tray reactors as described in PCT/US2017/020050.

Example 2. Growth of Fusarium oxysporum Strain MK7 Biomat on NutrientMedium Refreshed Daily (Semi-Static Conditions)

Dense Fusarium oxysporum strain MK7 biomats approximately 3 cm thickwere grown in 21 days on nutrient medium that was refreshed daily. Thebiomats were generated using sterile MK7-1 liquid medium (described inPCT/US2017/020050) containing 7.5% glycerol at pH 3.0 in 12.7×17.8 cmPyrex® glass trays. To initiate the experiment, 200 mLs of the nutrientmedium was inoculated with 5% (volume/volume) of Fusarium oxysporumstrain MK7 culture in the late exponential growth phase as describedpreviously in PCT/US2017/020050. 200 mLs of the inoculated medium wereadded to each of three sterile trays that were lined with sterile coarsenylon mesh screens. The cultures were incubated undisturbed for 4 daysat room temperature (˜22° C.) to allow development of the initial biomatlayer that formed at the surface of the liquid. After 4 days of growth,the biomats were gently lifted out of the tray using the nylon meshscreens and were tilted at a 45 degree angle to allow the liquid todrain out of the mats. The biomats were allowed to drain in thisposition until less than one drop of liquid dripped out every fiveseconds. Sufficient draining occurred, on average, after about 3minutes. The drip-dried biomats in their screens were placed in freshpreweighed 12.7×17.8 cm Pyrex® trays containing 200 mL of freshMK7-glycerol medium (described in PCT/US2017/020050). Trays with biomatswere re-weighed. The process of moving the biomats to another traycontaining fresh medium was repeated on approximately a daily basis for17 more days. Sampling of one of the biomats occurred on days 12, 15 and21 and the moisture contents of these biomats were determined. Theaverage moisture content of the biomats was 17.3% (std dev=0.7) and thisvalue was used to calculate dry biomass production over the duration ofthe experiment. Dry biomass production was linear from day 4 through day18 (r²=0.995) after which biomass weight stabilized at about 2.5 Kgdry/m² (FIG. 1, y-axis normalized to a per m² basis, growth is typicallyexponential between day 0 and day 4). The average growth rate over thistime period of linear growth was 6.04 g/m²/h. FIG. 2 shows a ˜3 cm thickbiomat that developed after a total of 21 days growth using this method.

Example 3. Growth of Biomats Under Continuous Flow Conditions

A continuous flow bioreactor system was fabricated to demonstrate growthof biomats on the surface of flowing liquid media. The system wasfabricated from a 2.44 m long clear plastic roofing panel with a seriesof corrugations that were used as flow channels (FIG. 3). The ends ofeach of the channels were dammed with silicon (100% Silicone, DAPProducts Inc., Baltimore, Md.) enabling liquid to be retained within thechannels. Flow was facilitated through the channels by delivery ofliquid media to one end of the channels via a peristaltic pump, with theliquid exiting the other end of the channels through holes in the bottomof the channels. The whole plastic roofing panel system was slanted atan angle of 1 cm rise per 1 m run to enable about 500 mL of liquid to beretained in each channel and a consistent flow being a function of theamount of liquid and the angle of the inclination.

The panel system was sanitized and wrapped in Saran®-like plastic wrapto isolate the system from the surrounding room environment. Sterile airwas pumped under the plastic wrap at a rate of 400 mL/min creating apositive pressure on the system. To initiate development of a biomatprior to starting flow, a 500 mL volume of nutrient medium inoculatedwith the desired filamentous fungus was added per channel and allowed toincubate under quiescent/static conditions for 4 days. After 4 days, theperistaltic pump delivered a continuous pulsed flow of 400 mL/d to“feed” the biomats (ON at 2.016 mL/min for 49 min, 39 sec; OFF for 5 h10 min 21 sec). Two independent experiments were conducted with eachexperiment using two separate flow channels as replicates (FIG. 3).

Consolidated biomats were harvested after 10 days of growth on thenutrient medium (4 days under quiescent/static conditions followed by 6days under continuous flow; FIG. 4). Average dry weight of the producedbiomass was an average of 2.38 g for the replicate flow channels. Duringthe continuous flow periods (day 4 to day 10) the average removal ratesof C and N from the flowing liquid medium by the growing biomats were11.9 and 1.2 mg/L/h, respectively. C and N removal rates from the liquidmedium were determined by measuring liquid volume and total C and Ninputs and outputs from the bioreactor system using a Costech total Cand N analyzer (ECS 4010, Costech Analytical Technologies, Valencia,Calif.). Thus, the continuous flow system supported biomat growth at thesurface. The experiments also served as a laboratory-scale demonstrationfor continuous feed of Fusarium oxysporum strain MK7 biomat growth andproduction of consolidated biomats. It should be noted that otherfeedstocks, flow rates and resulting growth rates can be achieved withthis type of system. For example, with 10% glycerol in MK7-1 medium(described in PCT/US2017/020050) at pH 2.8, expected yields are greaterthan 40 grams dry biomass per day per m².

Example 4. Semi-Continuous and Continuous Production of Fusariumoxysporum Strain MK7 Biomats

Dense Fusarium oxysporum strain MK7 biomats were grown and harvested ona semi-continuous basis over a period of 19 days. The biomats weregenerated using acid whey as the feedstock/carbon source supplementedwith ½ strength MK7-1 medium salts (described in PCT/US2017/020050)adjusted to pH 4.0. To initiate the experiment, 200 mL of the nutrientmedium inoculated with Fusarium oxysporum strain MK7 (5% volume/volume)in the late exponential growth phase was added to sterilized 12.7×17.8cm Pyrex® glass trays, which were then covered with Saran® wrap andincubated at room temperature. After 5 days of growth, ⅓ of the biomatfrom one end of the tray was removed by cutting and removing a 5.9×12.7cm section of biomat (FIG. 5A). The remaining ⅔ of biomat was thenphysically moved over to the open area of medium that was created byremoval of the ⅓ portion of biomat. The biomat was shifted by physicallygrasping it with sterile gloved fingers and pulling the biomat overuntil it touched the end of the tray to open medium with no formedbiomat at the other end of the tray (FIG. 5B). The process of harvestinga ⅓ section of the most mature portion of the biomat and then moving theremaining ⅔ of biomat over the open area was repeated periodically. 50mLs of medium were aseptically removed from the tray every 4 days andreplaced with 50 mLs of fresh sterile medium (acid whey with ½ strengthMK7-1) to replenish the nutrients removed from the liquid medium byremoval of the biomat. Dry biomass production using this method yielded0.57 g/day per tray or 25.2 g/d/m² between days 5 and 19 (FIG. 6). Thus,a semi-continuous production system was demonstrated whereby the mostmature end of the biomat was harvested at an average rate of 1.56 cm/dayand fresh biomat growth was initiated in the open area of medium at theother end of the tray.

The system is also amenable to continuous harvesting and growth of abiomat whereby continuous removal is facilitated by a roller wheel thatis attached to the mature end of the biomat (FIG. 7). The roller wheelslowly turns and harvests the mature biomat and at the same time createsan open medium for growth of new biomat at the other end of the tray.The roller wheel turns and harvests the biomat at a rate of 1.56 cm/dayto reproduce the semi-continuous system described above. It is desirablethat the nutrients in the liquid medium be replenished at the rate ofnutrient removal by the biomat.

Example 5. Membrane Encapsulated Bioreactors

Dense Fusarium oxysporum strain MK7 biomats were grown in liquid growthmedium that was encapsulated in a bioreactor system with no gasheadspace. Sterile Petri dish bottoms (55 mm diameter) were filled tothe brim with 57 mL of inoculated MK7-1 medium (described inPCT/US2017/020050) containing 8% glycerol. Gas permeable/semi-permeablemembranes of polypropylene and polycarbonate were placed directly on thesurface of the liquid medium and sealed tightly with rubber bands. Nogas headspace was provided at the start of the growth period.

After inoculating the medium and sealing the membranes, the bioreactorswere allowed to sit undisturbed until harvest. FIG. 8 shows the ˜5 mmand ˜1 mm thick biomats of Fusarium oxysporum strain MK7 that grewdirectly underneath the polypropylene (FIG. 17A-C) and polycarbonate(FIG. 17 D) membranes in five days, respectively. The biomats mildlyadhered to the membranes and could be easily harvested by simply peelingaway the biomats from the membranes (FIG. 17).

Example 6: Production of Pigments and Vitamin D2 by Irradiation ofFusarium oxysporum MK7 Biomats with UVB

UVB light (290-320 nm) was used to trigger pigment production byFusarium oxysporum strain MK7 biomats. Fusarium oxysporum strain MK7biomats produced in 3 days on 7.5% glycerol MK7-1 medium (described inPCT/US2017/020050) were irradiated with UVB light for a period of 4hours. The UVB light was emitted from a 50 W bulb (Slimline Desert 50UVB T8 fluorescent bulb, 46 cm; Zilla, Franklin, Wis.) placed 10 cmabove the biomat. Orange pigmentation was visually detected after 0.5 hof irradiation and was pronounced after 4 h of irradiation (FIG. 9). Inaddition, biomats that have not been exposed to UVB light have a vitaminD2 content of less than 50 IU/100 g of biomat whereas after UVB lightexposure for approximately 12 hours the vitamin D2 content is increasedto approximately 1.2 million IU/100 g biomat.

Example 7: Fusarium oxysporum Strain MK7 Biomats Grown on a Mixture ofGlycerol, Starch and Corn Steep Liquor

Fusarium oxysporum strain MK7 biomats were produced from a mixture ofglycerol, starch, corn steep liquor and MK7-1 salts (described inPCT/US2017/020050) in as little as 4 days. Glycerol was purchased fromDuda Energy LLC (Decatur, Ala.; 99.7% Purity; USP Grade; Lot#466135376340); 100% Argo Corn Starch manufactured by Argo FoodCompanies, Inc (Memphis, Tenn.) was purchased from Albertson'ssupermarket in Bozeman, Mont., and the corn steep liquor was purchasedfrom Santa Cruz Biotechnology, Inc. (Dallas, Tex.; Lot #B0116). Thegrowth medium was a mixture of 7.5% glycerol (weight/weight), 2.5%starch and 2.5% corn steep liquor with MK7-1 salts. The mixture wasadjusted to pH 3.3 by adding an appropriate amount of HCl and boiled for15 minutes in a suitable container. After cooling to room temperature,the pH of the mixture was readjusted to 3.3 and then inoculated with 5%Fusarium oxysporum strain MK7 inoculum (vol/vol) as prepared inPCT/US2017/020050. Aliquots of 1.5 L inoculated media were added tothree sanitized 0.25 m² polypropylene trays, placed in a sanitized trayrack system that was completely covered with aluminum foil to createdark conditions, and incubated at 23°±1° C. The filamentous fungalbiomats that grew at the surface of the medium were harvested after 4days by simply lifting the biomats from the trays.

The average final pH of the residual liquid in the three trays was 4.45(standard deviation=0.14). Three 56.7 cm² circular portions were cut outand removed from each of the biomats at random positions and theseportions were dried at 50° C. for 48 h to obtain dry weights. Theaverage biomass dry weight (standard deviation) was 124.6 g/0.25 m²(43.4) or 498.4 g/m² (173.6). The mean thickness of the moist biomatswere 7.5 mm and the mean density on a dry weight basis was 0.66 g/cm³.

To expose the biomat filaments and enable examination by Field emissionscanning electron microscopy (FE-SEM), the extracellular polymericsubstances (EPS) between the filaments were removed by washing withethanol. To accomplish this, 1 cm² portions (1 cm×1 cm) of the biomatswere excised with a razor blade immediately before harvesting, and theexcised portions were subjected to an ethanol washing/dehydration seriesby sequentially submersing the samples for the noted times in 40 mL ofthe ethanol mixtures as follows: 25% ethanol, 75% deionized H₂O for 20minutes; 50% ethanol, 50% deionized H₂O for 20 minutes; 75% ethanol, 25%deionized H₂O for 20 minutes; 95% ethanol, 5% deionized H₂O for 20minutes; 100% ethanol, 0% deionized H₂O for 60 minutes. The 100% ethanoltreatment was repeated 2 more times before storing the samples in 100%ethanol.

To retain microstructure integrity of the biomats for FE-SEM, ethanolwashing/dehydration was followed by critical point drying using aTousimis Samdri-795 critical point dryer according to the manufacturerinstructions (Tousimis Samdri-795 Operations Manual; Tousimis,Rockville, Md.). After critical point drying, the samples were eithermounted directly onto aluminum stubs or sliced into <0.3 mm thicksections with a razor blade prior to mounting. The samples were thencoated with iridium (20 μm, EMITECH K575X, Electron Microscopy Sciences,Hatfield, Pa.) and examined with a JEOL 6100 FE-SEM using an incidentbeam energy of 1 keV (JEOL USA, Inc., Peabody, Mass.).

FE-SEM imaging revealed a complex network of interwoven hyphal filaments(FIG. 10), very similar to the structure revealed by light microscopyfor biomats grown on glycerol as reported in PCT/US2017/020050. Threedistinct layers were observed: (a) an aerial hyphae layer at the topsurface, (b) a dense bottom layer and (c) a transitional layer betweenthe top and bottom layers. The transitional layer was only looselyattached to the dense bottom layer, thus enabling easy separation of thebottom layer from the rest of the biomat. Filament densities of thetransitional layer ranged from slightly less dense than the bottom layerin the zone where the two layers met, to a density that was comparableto the aerial hyphae near the top of the biomat.

Excised samples were also prepared for light microscopy by slowlydipping into the following solutions in the order and times shown below:

Xylene, 3 min; Xylene, 3 min; 100% ethanol, 3 min; 100% ethanol, 3 min;95% ethanol, 3 min; 95% ethanol, 3 min; 70% ethanol, 3 min; Deionizedwater, 3 min; Hematoxylin 1, 1.5 min; Running tap water rinse, 1 min;Clarifier solution, 1 min; Running tap water rinse, 1 min; Bluingsolution, 1 min; Running tap water rinse, 1 min; 70% ethanol, 30 dips;95% ethanol, 30 dips; 95% ethanol, 30 dips; 100% ethanol, 30 dips; 100%ethanol, 30 dips; 100% ethanol, 30 dips; Xylene, 30 dips; Xylene, 30dips; Xylene, 30 dips; Apply cover slip.

The above procedure was followed by visualization with a lightmicroscope (B400B, Amscope, Irvine, Calif.) at 100× magnification (FIG.11).

Sections of the biomats approximately 2 cm² in size were excised fromthe fresh biomats with a razor blade immediately before harvesting.These sections and then immersed in 35 mL of deionized water in 50 mLconical bottom centrifuge tubes. The tubes were sonicated (CP200TUltrasonic Cleaner, Crest Ultrasonics, Ewing, N.J.) for either 0, 40, 90or 150 seconds to disperse filaments into the liquid and enablemicroscopic observation. Aliquots of the liquid (˜100 uL) from thesetubes were placed on a glass slide, covered with a cover slip andobserved with a light microscope (B400B, Amscope, Irvine, Calif.) at100× magnification. The average length (std dev) of non-broken filamentswere measured and determined to be 1.1 (0.6), 1.2 (0.4), 1.0 (0.4) and1.2 (0.2) mm for the 0, 40, 90 and 160 second sonication treatments,respectively. The maximum filament length observed in each treatmentwere 2.5, 1.4, 1.8, and 1.4 mm, respectively. These filament lengths aresignificantly longer compared to growth of Fusarium oxysporum strain MK7in submerged shake flask cultures where average lengths are less than0.02 mm.

Example 8: Production of Chicken Nuggets Using Fusarium oxysporum StrainMK7 Biomats Grown on a Mixture of Glycerol, Starch and Corn Steep Liquor

Fusarium oxysporum strain MK7 biomat, produced as described above, wereused to create chicken nuggets. Moist biomats were steamed in a potsteamer at 97° C. for 0.5 hour, cooled to room temperature and used asthe base to produce chicken nuggets. Steamed moist biomat (200 g) waschopped into pieces less than 0.5 mm long and homogenized with 4%(weight/weight; 8 g) chicken base and 4% egg white protein (8 g). Theresulting mixture comprised more than 90% Fusarium oxysporum strain MK7biomat. Portions of this biomat mixture (˜30 g) were formed into nuggetshapes and steamed for in a pot steamer. The prepared nuggets werebreaded by coating in egg whites and then mixing with bread crumbs thatadhered to the surface prior to frying. The prepared nugget exhibited achicken meat like texture (FIG. 13A) and exuded the typical aroma ofchicken. Taste testing by 5 people deemed the nugget to closely simulateactual chicken containing chicken nuggets in terms of taste and texture.

Example 9: Production of Fusarium oxysporum Strain MK7 Biomat Extract

Highly concentrated and viscous extracts were produced from Fusariumoxysporum strain MK7 biomats. Biomats harvested after 4-16 days ofcultivation, as previously described, are rinsed and steamed, drip driedon porous plastic mesh for 5 minutes, and placed in plastic bags andsealed. Sealed bags are frozen at either −20° C. or −80° C. for 24 hoursprior to being incubated at 60° C. incubator in the original sealed bagsfor 48 hours after pH adjustment of the remaining medium liquid tobetween pH 4-6. After heat treatment, biomats are pressed through <1.5mm pore size filters and the resulting liquid collected. The collectedliquid is boiled for 10 minutes in a non-reactive vessel then dried at60° C. until water content is ˜6-8%, forming a sticky paste extract. Thenutritional value of the extract is similar to the nutritional value ofthe steamed biomat and flour made from steamed biomats.

Example 10. Production of Yogurt from Fusarium oxysporum Strain MK7Biomats Grown on Acid Whey

Fusarium oxysporum strain MK7 biomats were used directly to produceyogurt. The biomats were grown in trays on an acid whey feedstock/carbonsource that was generated as a by-product of Greek yogurt manufacture,harvested after 6 days and were steamed within 20 minutes of harvesting.200 g of the cooled, moist biomass was blended together with 600 g ofdrinking quality tap water to produce a milk-like suspension referred toas “MK7 liquid dispersion.” The MK7 liquid dispersion was used as aningredient by itself or in combination with cow's milk to produceyogurt.

Three mixtures containing different ratios of MK7 liquid dispersion towhole milk were prepared: 1) 25% MK7 liquid dispersion:75% whole milk,2) 50% MK7 liquid dispersion:50% whole milk, and 3) 100% MK7 liquiddispersion. The mixtures were used to make three batches of yogurt byheating each mixture to 83° C. and holding at that temperature for 14minutes with constant stirring. The mixtures were allowed to cool to 43°C. and then live yogurt cultures added as inoculum. The resultingmixture was incubated at 44° C. in a yogurt maker (Model YM80;EuroCuisine, Los Angeles, Calif.) for 8 hours. All of the resultantmixtures had the appearance and texture of yogurt (FIG. 14), as well asa smell and taste similar to typical yogurt.

Example 11: Growth of Mushroom Biomats on Glycerol

Biomass biomats comprised of Baby Bella Brown Crimini Mushrooms(Agaricus bisporus) and White Mushrooms were produced in as little as 10days using glycerol as the primary carbon source (feedstock). Thesecommon edible mushrooms were purchased from Albertson's supermarket inBozeman, Mont. and stored at 4° C. The medium used to grow the mushroomsconsisted of 1 L of 7.5% glycerol with MK7-1 salts (described inPCT/US2017/020050) that was boiled for 10 minutes followed by cooling toroom temperature (˜23° C.). The pH of the mixture was adjusted to 2.7and 200 mL of the pH adjusted mixture was poured in two sterile12.7×17.8 cm Pyrex® trays. The inoculum consisted of 5 g of blended,surface-sterilized Crimini or White Mushrooms that was added to themedium in each tray. The mushroom inoculum was prepared as follows: 1)10 g of moist Crimini or White Mushrooms were added to 200 mL of a 5%bleach solution and the suspension was stirred for 2 minutes to surfacesterilize the mushrooms, 2) the mushrooms were then rinsed bytransferring into 200 mL of sterile glycerol/MK7-1 salts medium(described in PCT/US2017/020050) and stirring for 2 minutes, 3) thesurface sterilized mushrooms were blended for 30 seconds in a coffeegrinder that had been sterilized by rinsing with 70% ethanol, 4) theground mushroom biomass (<5 mm long aggregates) was surface sterilizedagain by repeating steps 1 and 2 with the ground biomass, 5) 5 grams ofthe ground mushroom biomass was added to the liquid medium in the Pyrex®trays (final pH=4.0-4.1 after addition of mushrooms), and 6) the trayswere covered and allowed to incubate at room temperature (22±2° C.) inthe dark.

Biomats were observed to develop on the surface of the medium after 3days of incubation and consolidated biomats were harvested after 10 daysof growth. Biomats of Crimini Mushrooms covered the entire surface ofthe liquid medium in the tray while biomat growth of White Mushroomscovered approximately ½ the liquid medium as five floating biomatislands. The mean thickness of the biomats were 1.5 mm for the Criminiand 1.7 mm for the White Mushrooms. Biomass biomats were dried at 50° C.for 48 h and the dry weights produced per tray were 1.14 g and 2.12 gfor the Crimini and White Mushrooms, respectively. Densities on a dryweight basis for the dry biomass biomats were 0.033 and 0.111 g/cm³ forthe Crimini and White Mushrooms, respectively.

Microscope images revealed the mycelial nature of the biomats. Averagehyphal thicknesses were 25.2 μm (std dev=6.2) and 18.7 μm (4.0) for theCrimini and White Mushroom biomats, respectively.

Produced Crimini biomats were used to create chicken nuggets. Biomatswere steamed at 97° C. for 0.5 hour, cooled to room temperature and usedas the base to produce chicken nuggets. Steamed moist biomass (2.5 g)was mixed with 3% (weight/weight; 75 mg) Better Than Bouillon chickenbase (Southeastern Mills, Inc. Rome, Ga.) and 3% Eggwhite Protein (75mg; Now Foods, Bloomingdale, Ill.) and chopped into pieces less than 2mm long using a razor blade. The mixture was formed into a nugget andsteamed for 0.5 hour. The prepared nugget provided the typical aroma ofchicken with a slight mushroom fragrance. When tasted, the nugget had achicken to neutral flavor.

Example 12. Growth of Mushroom Biomats on Malt and Glycerol Media

Biomass biomats comprised of Calvatia gigantean (giant puffball),Pleurotus ostreatus (pearl oyster), Pleurotus ostreatus var. columbinus(blue oyster), Hypsizygus ulmarius (elm oyster), Sparassis crispa(cauliflower) and Ganoderma lucidum (reishi) were produced in as littleas 5 days using Malt Extract Medium 001, Glycerol Medium 002, Hansen'sMedium, MK7-SF Medium, Malt Extract+NH₄NO₃ Medium 003 (Table 3). Allfinal media contained 0.01% chloramphenicol.

TABLE 3 Ingredients added to deionized or drinking quality tap water toprepare nutrient media. Malt Extract Medium 001 Ingredient Amount GradeLot # Vendor Location Light Pilsner  40.0 g Food 180526B Homebrewstuff,com Boise, ID Malt Peptone  4.0 g Research 44984- Research Products Mt.57374 International Prospect, IL Yeast Extract  1.2 g Research 53852-Research Products Mt. Powder 66581 International Prospect, IL Canola Oil 1.0 mL Food SEP/25/19 Better Living LLC Pleasanton, CA S3283 CA GroundOats  4.0 g Food Jan. 25, Walmart-Stores, Inc Bentonville, 2020 I2M AR06:36 Tap H₂O  1000 mL N/A N/A N/a Bozeman, MT Glycerol Medium 002Ingredient Amount Grade Lot # Vendor Location Glycerol  40.0 g Food/USP20149018137001 Duda Energy LLC Decatur, AL Peptone  4.0 g Reagent44984-57374 Research Products Mt. International Prospect, IL YeastExtract  1.2 g Reagent 53852-66581 Research Products Mt. PowderInternational Prospect, IL Canola Oil  1.0 mL Food Sep. 25, 2019 CABetter Living LLC Pleasanton, S3283 CA Ground Oats  4.0 g Food Jan. 25,2020 I2M Walmart-Stores, Bentonville, 06:36 Inc AR Tap H₂O  1000 mL N/AN/A N/a Bozeman, MT Hansen's Medium Ingredient Amount Grade Lot # VendorLocation Peptone  1.0 g Reagent 44984-57374 Research Products Mt.International Prospect, IL KHPO4 *  0.3 g Reagent Mfg. Doesn't useEisen-Golden Dublin, 7H₂O lot numbers Laboratories CA MgSO4 *  2.0 g USP81721 San Francisco Salt San 7H₂O Co. Leandro, CA Glucose  5.0 g Reagent0435C235 Fisher Scientific Denver, CO Tap H₂O  1000 mL N/A N/A N/aBozeman, MT MK7-SF Medium Ingredient Amount Grade Lot # Vendor LocationNH₄NO₃ 7.553 g ACS A0390194 Acros Organics Somerville, NJ Urea 2.548 gUSP 30570-67229 Research Products Mt. International Prospect, IL CaCl₂2.000 g Reagent 102615 Fritz Pro Aquatics Mesquite, TX MgSO₄ * 2.000 gUSP 81721 San Francisco Salt San 7H₂O Co. Leandro, CA KH₂PO₄ 7.500 gReagent Mfg. Doesn't use Eisen-Golden Dublin, lot numbers LaboratoriesCA Trace * 2.000 mL * * * * Glycerol 0.075 Kg Food/USP 20149018137001Duda Energy LLC Decatur, Yeast Exract 1.750 g Research 53852-66581Research Products Mt. International Prospect, IL FeCL₂ * 4H₂O 0.020 gReagent 951164 Fisher Scientific Fair Lawn, NJ DI H₂O 0.940 L N/A N/AN/A Bozeman, MT Trace Components * Micronutrients * mg/L Grade Lot #Vendor Location FeSO4•7 H2O  9.98 ACS 3562C398 Amresco Solon, OH ZnSO4•7H2O  4.4 USP/FCC 61641 Fisher Waltham, MA MnCl2•4 H2O  1.01 Reagent13446-34-9 Fisher Waltham, MA CoCl2•6 H2O  0.32 Reagent 7791-13-1 FisherWaltham, MA CuSO4•5 H2O  0.31 Technical 114675 Fisher Waltham, MA(NH4)6Mo7O24•4  0.22 ACS 68H0004 Sigma St. Louis, MO H2O H3BO3  0.23 ACS103289 Fisher Waltham, MA EDTA, free acid 78.52 Electrophoresis 46187Fisher Waltham, MA Mat Extract + NH4NO3 Medium 003 Ingredient AmountGrade Lot # Vendor Location NH4NO3  5.0 g ACS A0390194 Acros OrganicsSomerville, NJ Light Pilsner  40.0 g Food 180526B Homebrewstuff, comBoise, ID Malt Peptone  4.0 g Research 44984- Research Products Mt.57374 International Prospect, IL Yeast Extract  1.2 g Research 53852-Research Products Mt. Powder 66581 International Prospect, IL Canola Oil 1.0 mL Food Sep. 25, 2019 Better Living LLC Pleasanton, CA S3283 CAGround Oats  4.0 g Food Jan 25, Walmart-Stores, Inc Bentonville, 2020I2M AR 06:36 Tap H₂O  1000 mL N/A N/A N/A Bozeman, MT

The above recipes in Table 3 were used to prepare media in either 2 LPyrex® bottles or 8 L stainless steel pots by mixing the specifiedingredients into the specific volumes of water depending on the volumeof media desired. Ingredients were added to water while liquid wascontinuously stirred with a stir bar or a spoon. Each component of themedia was thoroughly mixed into the liquid before the next component wasadded, pH for the MK7-SF medium was adjusted to 5.0, and the solutionsautoclaved. All other pH's resulted from simply mixing the ingredients.The medium and vessels were autoclaved for at least 20 minutes at 20 psiand 121° C. Osmotic pressure of the liquid was measured using anAdvanced Instruments, Inc. osmometer Model 3250 (Two Technology Way,Norwood, Mass.).

After autoclaving, the media were allowed to cool to room to temperatureand individual vessels were inoculated with the mushroom species shownin Table 4.

TABLE 4 Mushroom spores (10 cc syringes) were purchased from MycoDirect(12172 Route 47, Ste 199 Huntley, Il 60142) and received on Aug. 2,2018. Elm Oyster spores were purchased from Everything Mushrooms (1004Sevier Ave Knoxville, TN 37920) and received on Aug. 3, 2018. Lot DateProduced by Company Blue Oyster 3-P7 February 2018 Pearl Oyster 9P8December 2017 Giant Puffball N/A March 2018 Cauliflower Mushroom N/AApri 2018 Elm Oyster (Ice dried) N/A October 2017

Inoculation of growth media was preformed using the following methodsapplied using aseptic technique. All aseptic work in these experimentswere performed in Class II biosafety cabinet. Spore syringes were usedto directly inoculate approximately 75 mL of growth medium in previouslyautoclaved, 12.7×17.8 cm Pyrex® glass trays. This was done byaseptically transferring liquid medium into an autoclaved Pyrex® trayand inoculating with 2 cc of the suspension contained in the sporesyringe. The tray was covered with sterile aluminum foil and then gentlyswirled to mix the inoculated medium.

Malt Extract Agar (MEA; Table 5) plates were prepared aseptically byautoclaving MEA, allowing to cool to 50° C., and pouring ˜25 mL into100×15 mm sterile Petri dishes.

TABLE 5 Ingredients used to prepare Malt Extract Agar Malt Extract Media(MEA) Ingredient Amount Grade Lot # Vendor Location Light Pilsner   30.0g Food 180526B Homebrewstuf.com Boise, ID Malt Agar   20.0 gMicrobiological 2170501 BD Sparks, MD Tap H₂O  1000 mL N/A N/A N/ABozeman, MT

MEA plates were inoculated by aliquoting 1 cc of liquid from thesuspension contained within the spore syringe onto the plates. The agarplates were then sealed with Parafilm® and placed into a clean darkdrawer at room temperature.

After mycelium had covered the entire surface of the MEA plates, theywere used for inoculation of 1.5 L medium in 2 L baffled shaker flasks.Approximately 2 cm² portions of agar medium with mycelium on the surfacewere excised from the plates with a sterile razor blade and diced into˜2 mm² portions, which were then added to two flasks containing 1.5 L ofMalt Extract 001 medium. The medium was incubated for 3 days at roomtemperature (23±1° C.) with intermittent shaking by hand (flasks werevigorously shaken by hand for 1 minute at a minimum of five times perday).

The cultures in the shaker flasks were then used as inoculum for 6 L ofMalt Extract medium 001 and for 6 L of Malt Extract+NH₄NO₃ 003 medium.The media were inoculated with 15% (vol:vol) of inoculum culture andmixed thoroughly. Two liters of inoculated media were poured into eachof three 0.25 m² plastic trays that were placed into a tray rack. Theracks were wrapped in Saran® and allowed to incubate for 6 days.Relatively dense biomats covering the entire surface within 4 days andthe biomats were harvested after 6 days.

Biomats from 12.7×17.8 cm Pyrex® glass trays and the 0.25 m² plastictrays were harvested by lifting the biomats from the trays and gentlysqueezing by hand. Portions of the biomats (3-50 g) were streamed for 20minutes over boiling water (˜5 cm above surface of water) in a potsteamer set on a kitchen oven burner. After steaming, the biomass wasallowed to cool to room temperature and immediately bagged in a Ziploc®bag and sent to Eurofins (Des Moines, Iowa) for protein analysis (N bycombustion, Test Code QD252).

TABLE 6 Results from a series of Giant Puffball growth in trays invarious types of media Biomass Tensile Final per Strength Tray pH IonicOsmotic pH Surface of Wet Size Initial Strength Pressure Time FreeProtein Area Density Biomat Media (m²) Media C:N (mmol/L) (mOsm) (days)Liquid (%) (g/m²) (g/cm³) (g/cm³) Malt 0.022 6.28 19 33.1 169 5.7 5.6232.03 71.4 0.057 314.1 001 Glycerol 0.022 6.96 30 13.6 505 5.7 5.54 N/A40 0.04 214.9 002 Hansen's 0.022 8.81 27 30.7 39 N/A N/A N/A N/A N/A N/AMK7-SF 0.022 4.91 7.5 344 1387 9.0 5.07 46.33 178.6 0.045 135.0 Malt0.25 6.96 19 33.1 169 6.2 6.25 32.04 111.1 0.037 264.0 001 Malt + 0.256.88 7.5 145.1 287 5.8 N/A 46.88 108.3 0.11 281.1 NH₄NO₃ 003

Example 13. Fusarium oxysporum Strain MK7 Chicken Nugget

Chicken flavored Fusarium oxysporum strain MK7 is a basic ingredient toa number of recipes including chicken nuggets, with or without breading,chicken for Asian dishes, or other chicken dishes as a chickenreplacement. Fusarium oxysporum strain MK7 biomats produced fromdifferent feedstocks/carbon sources result in slightly different chickenflavors. The glycerol chicken is sweeter and the acid whey chicken tendsto be a little bit sourer.

The amount of food processing and the blade used (i.e. sharp metalblade, dull metal blade, plastic blade) result in different chickennugget textures. Further, acceptable chicken nuggets can be producedfrom a wide variety of biomass sizes. That is, biomass can be cut with aknife, lightly food processed or highly food processed and still resultin acceptable chicken analogs.

A 50-20:1:1 ratio of Fusarium oxysporum strain MK7:chicken stock:binderwas used with or without approximately a 66.6% Fusarium oxysporum strainMK7:fat ratio. Suitable fats include duck fat, coconut butter, and cocoabutter. After mixing, the mixture is steamed for approximately 30minutes to set the binder; however, some binders may require more orless time. Additional breading can then be added and the resultingnuggets process as typical for such foodstuffs.

Example 14: Breakfast Sausage and/or Hot Dog and/or Burger

An appropriate spice mix is added to size reduced Fusarium oxysporumstrain MK7 biomats as needed to develop the flavors desired, which maybe between 10 wt. % of spice mix to a quantity of Fusarium oxysporumstrain MK7 up to 20%, oftentimes in a ratio of 10 Fusarium oxysporumstrain MK7:1 spice mix, with or without additional ingredients such asonion, binders, and a fat such as cocoa butter. The mixture is thenfried to remove an appropriate amount of moisture. Additionalingredients can then be added, such as bulgur, vegetable broth,potatoes, etc. prior to shaping in the desired shape and cooking.

Example 15: Ice Cream and Mousse

A ratio of approximately 1:3 Fusarium oxysporum strain MK7 biomat:wateris generated having a particle size with average filament lengths lessthan 900 microns. This mixture is gently heated until there is no longera fungal scent and then used in approximately a 4:1 ratio with cashews,optionally with an appropriate amount of xanthan gum and/or flavoring,to generate a mix which may be optionally heated and then cooled to forma mousse. For frozen dessert, the mix is then placed in an ice creamchurner and, after churning, frozen to form a non-meltable frozendessert (FIG. 14).

Example 16: Production of Truffle Oil from Truffle Biomats

Oil extract can be prepared from Truffle (Tuber sp.) biomats grown asdescribed above. In one instance, truffle biomats were grown in trays inas little as 7 days using malt extract, glucose and peptone as theprimary carbon sources (feedstock). The edible Truffle mushroom waspurchased from IGB Trading LLC on the Amazon Marketplace and stored at4° C. A pure culture of the Tuber sp. fungus was prepared from thepurchased truffle by placing ˜3 mm³ portions of truffle (cut with asterile razor blade) on Malt Extract Agar+0.01% chloramphenicol (used toinhibit bacterial growth). A Malt Extract Agar was prepared by mixing 20g of malt extract, 20 g of glucose, 1 g peptone and 20 g of agar in 1 Lof deionized water prior to autoclaving for 30 minutes and cooling to50° C. before adding 0.01% chloramphenicol. The sterile mixture was thenpoured into 9 cm diameter Petri plates and allowed to cool and solidify.

The fungus was observed to grow on the trays after 3 days. After 4 daysof growth, hyphae were picked with a sterile microbiological loop andstreaked onto a fresh set of Malt Extract Agar+chloramphenicol plates.The fungus was allowed to grow on said plates for 5 days, after whichhyphae were picked with a microbiological loop and used to confirmculture purity by DNA sequencing. Confirmation was accomplished byextracting and purifying the DNA (FastDNA Spin Kit, MP Biomedicals) andsequencing the ITS region of the metagenome followed by phylogeneticclassification of the sequences using Blast (NCBI database).

Malt Extract Broth was prepared by mixing 20 g of malt extract, 20 g ofglucose and 1 g peptone in 1 L of deionized water and sterilized.Scrapes of the hyphae with the microbiological loop were also used toinoculate 50 mL of sterile Malt Extract Broth in sterile baffled shakerflasks capped with sterile gauze material. Sterile gauze was used as itallowed exchange of gases into and out of the shaker flask. Shakerflasks were then rotated at 185 rpm for 5 days. The rotated cultureswere then used to inoculate 350 mL of sterile Malt Extract Broth insterile 12.7×17.8 cm Pyrex® glass trays. The inoculum density was forthis culture medium was 7.5% inoculum to 92.5% broth. After 7 days ofgrowth in the trays, the filamentous biomat formed on the surface washarvested by lifting the biomat from the liquid medium. The harvestedbiomats were dried at 40° C. for 48 h. Lipids/oil from these harvestedbiomats were extracted by either mechanical pressing or by solventextraction using hexane, although other extraction methodologies can beused. Can also use another extraction method Yuval will send.

Example 17: MK7 Flour

Fusarium oxysporum strain MK7 biomat, produced as described above, wasused to create dried powder similar in particle size and particle sizedistribution to a standard baking flour. Here, moist biomats weresteamed in a pot steamer at 97° C. for 0.5 hour, cooled to roomtemperature and dehydrated in a Cuisinart dehydrator (model DHR-20) for2-8 hours with an average dehydration time being 4 hours. Dehydrationtime is a function of the amount of biomass loaded into the dehydrator,distribution of biomats in the dehydrator which impacts air flow in thedehydrator and the water content of biomats (average water contentapproximately 75%) and room temperature. Water content post dehydrationvaries between 4 and 14% with average water content post dehydrationbeing below 12%. Dehydrated biomass was size reduced using a coffeegrinder (KRUPS, Electric coffee and spice grinder, stainless steelblades F2034251) until finely ground. Average particle size for groundbiomat flour ranged from 75 microns to 120 microns. A small fraction oflarger particles, app 5 wt %, had a particle size of greater than 180microns. A small fraction of smaller particles, app. 5 wt % had aparticle size smaller than 75 microns. Said smaller particles where offa size which enabled the small particles to remain air borne forextended periods of time. Particle size was determined by sifting 100gram samples of size reduced biomats for 5 minutes in sieves with 180μm, 120 μm and 75 μm openings. Water content post dehydration and postsize reduction below 6% is preferred as higher water contents can leadto clumping of dried and milled biomass.

Biomat flour was then used as an addition to other standard flours (KingArthur flour, Bob's Red Mill Flour & Bob's Red Mill Wheat Flour) and avariety of baked goods where prepared. Biomat flour was loaded at 5 wt%, 10 wt %, 20 wt % and 30 wt % with no deleterious effect on ultimatebaked good taste, rising, texture, appearance or smell. Productsdemonstrated included bread (7 grain, white & wheat), pastries (Pate aChoux), cookies, pasta and dumplings. The resulting products performedwell in taste tests and the inclusion of MK7 flour was not detectable tothose tasting the products.

Example 18: MK7 Extender

Fusarium oxysporum strain MK7 biomat, produced as described above, wasused to create particles of biomass that were used as an addition tomeat and fish as an extender (i.e. increase the amount of total foodproduct by the addition of MK7 to other exiting foodstuffs). Moistbiomats were steamed in a pot steamer at 97° C. for 0.5 hour, cooled toroom temperature. Biomats where size reduced (i.e. chopping with a knifeor food processing in a food processor) to a desirable particle sizedistribution. Size reduced biomass was then added to different foodproducts to extend the amount of meat in the case of a meat extender orfish in the case of a fish extender. As an example of meat extension.10%, 20%, 30%, 40% and 50% additions of size reduced biomass were addedto hamburger meat. Size reduction of biomass was evaluated at a numberof different size distributions. Smaller particle sizes tended toproduce denser and creamier textures. Larger particles tended to produceproducts with more texture, more mouth feel and required moremastication before swallowing. The extended meat was them processed asthough no biomass was added. In the case of hamburger extension, spicesor binders can be optionally added and the extended meat was formed intoa patty or meat ball and cooked until the meat was cooked to theconsumer desired temperature. Cooking methods included stove top, oven,frying and grill. Taste tests showed that acceptable food products whereproduced at all loading levels and all size distributions of addedbiomass. Chicken and pork extensions where also tried at similar loadinglevels with similar cooking and tasting results.

Fish extension was also demonstrated at 10%, 20%, 30% and 40% loadings.Fish fillet and fish balls where produced by adding processed MK7 at avariety of different size distributions ranging from small particles(less than 1.0 mm) to large particles (greater than 2 mm) with nodeleterious effect on taste, color, smell or over all eating experience.In the case of small particle size additions, resulting foodstuffs had acreamier texture. In the case of large particle size additions,resulting foodstuffs had a firmer texture characterized by largerparticles which required more mastication before swallowing. Taste testsshowed that acceptable food products where produced at all testedloading and size distribution levels.

Example 19: MK7 Jerky

Fusarium oxysporum strain MK7 biomat, produced as described above, wasused to create mycojerky, similar in appearance and taste to meatjerkies (i.e. beef jerky, buffalo jerky, pork jerky, chicken jerky,turkey jerky, etc.). Moist biomats were steamed in a pot steamer at 97°C. for 0.5 hour, cooled to room temperature. Biomats where size reducedto a size consistent with that normally found in jerky products. Sizereduced biomat pieces where in some cases seasoned for flavor anddehydrated in a Cuisinart dehydrator (model DHR-20) for 20-200 minuteswith an average dehydration time being 40-120 minutes. Dehydration timeis a function of the amount of biomass loaded into the dehydrator,distribution of biomats in the dehydrator which impacts air flow in thedehydrator, water content of biomats (average water contentapproximately 75%), room temperature and desired water content in thefinal product. Water content post dehydration varied between 8% and 12%depending on desired product characteristics. In some cases, perforatingthe biomass before dehydration produced a product that tore more readilyinto small pieces thereby easing consumption. Perforation of the biomasswas performed by using a fork, knife or tenderizer tool which bothperforated the biomass as well as disrupted the filament network suchthat it tore more easily. A large variety of spice mixtures (i.e. Cajun,cheese, soy, vinegar, herbs, sour cream & onion, liquid smoke, veganmeat flavors, etc.) where evaluated. Spice mixtures were evaluated bothbefore dehydration and post dehydration. Those samples which were spicedbefore dehydration offered more taste and better adhered to the biomassthan those which were treated after dehydration. The resulting jerkiesall performed well in taste tests.

Example 20: Myco-Chips

Fusarium oxysporum strain MK7 biomat, produced as described above, wereused to chips, similar in appearance and taste to potato chips or cornchips. Moist biomats were steamed in a pot steamer at 97° C. for 0.5hour, cooled to room temperature. Biomats where size reduced to a sizeconsistent with that normally found in chip products as well as highlyprocessed into a paste and formed into a chip like geometry. Myco-chipswhere then put into a frying pan of hot oil (temperature app equal to380° F.) until brown. Cooking times varied as a function of biomassgeometry but cooked very fast, usually in under 15 seconds. Producedfried chips proved to be very palatable and capable of offering a widevariety of taste experiences dependent upon spices added to or coatedupon the biomass pre-frying.

Example 21: Hermetically Sealed Bioreactor:Biomat

Pyrex® glass trays 12.7×17.8 cm as well as 100×15 mm Petri dishes areused as the base tray. The glass trays are loaded with 200 mL offeedstock mixed with liquid nutrient medium (if required) and inoculum.The trays are covered and sealed with the gas-permeable membrane that isattached to a plastic frame with an integrated rubber gasket. Thesealing system provides an effective aseptic seal between the membraneand the glass trays and enables easy assembly as well as opening/closingof the reactor for sampling and harvesting purposes.

A suite of different gas permeable membrane materials with different,thicknesses, pore sizes and textures (surface roughness) are used asmaterials for the gas liquid interface. Initially, eight (8) polymericmaterials are used including polypropylene, polyethylene,polytetrafluorethylene, polycarbonates, polyamides, polypyrrolones,poly(amidoamine) dendrimer composite and cellulose acetate (e.g.,Specialty Silicone Products, Inc. Corporate Technology Park, N.Y.;Thermo-Fisher, Waltham, Mass.; Merck Millapore, Burlington, Mass.).Three pore sizes are used for the materials (0.2, 0.45, 1.0 μm) thatfacilitate gas transfer in addition to the direct diffusion of gassesthrough the polymers themselves while excluding microorganisms.Additionally, sterile-cloth-like materials with different rough surfacetextures and tortuous paths for gas diffusion are used. A largeselection of such materials are commercially available from othercorporate sources including 3M, Solvay, Ube Industry and Saint-Gobain.

To analyze and determine parameters for different environmental andmission conditions, tray reactors are fitted with sensors to monitortemperature, dissolved oxygen and pH as a function of depth across thetray. Ports for sensors and wires crossing membranes into the reactorare sealed with silicone, epoxy, and/or adhesives depending on themembrane material. Septa integrated into the membranes are used assample ports for collecting liquid samples for analysis by GC-MS,ICP-MS, IC and total C/N. Standard as well as microelectrodes are usedto measure pH and electron acceptor flux (O₂) in real time across thegas-permeable membrane and within the biomat at regular time intervals(e.g., 6, 12, 24, 36, 48 hours). The flux information is important formatching real-time metabolic demands with membrane gas permeability andthe changing concentrations and distributions of electron donors(organics) and nutrients (inorganics) needed for optimal growth andfeedstock conversion.

Example 22: Un-Instrumented Reactors

Un-instrumented reactors used for growth studies with fungal strainFusarium oxysporum MK7 as a model filamentous organism. Strain MK7 is anextremophilic fungus that has been shown to thrive on a wide variety offeedstocks including human wastes, food wastes, cyanobacteria and algaebiomass, and lignocellulosic materials. Strain MK7 biomats have alsobeen shown to have tolerance to high urea levels (at least 26 g/L) aswell as high dissolved organic carbon, and osmotic pressure (300 g/Lglycerol).

The feedstocks tested include: 1) surrogate human urine as the primarysource of nitrogen; 2) surrogate food waste (dog food) as the primarycarbon source; and 3) plant material (lignocellulose) as an additionalcarbon source. All feedstocks are extensively analyzed for organic andinorganic constituents, pH, and biological oxygen demand. Surrogatehuman urine is prepared using a medium composition recommended by NASAscientists or other scientists involved in studying mission wastes.

The effectiveness of the different gas permeable membranes are measuredby conducting comparative biofilm-biomat growth studies whereindifferent membranes are sealed onto the surface of trays and Petridishes containing various feedstocks and MK7 inoculum. The membranes arein direct contact with the liquid phase and are the only avenue of gasexchange between the gas/vapor exterior environment and thebiofilm-biomats/liquid medium. Reactors are destructively sampled tomeasure growth (dry biomass weight, biomat thickness) over time. Growthrates are compared to control trays with no membranes. A factorialexperimental design consisting of feedstocks and membrane combinationsis tested to provide the best match of feedstock and membrane.Additional variables, including initial feedstock pH and inorganicnutrient additions, is also evaluated. Further, the experiments trackthe viable bacterial cell counts from feedstocks as a function of timeto quantify the disinfection kinetics linked to biomat growth.

Example 23

The best performing membrane/feedstock combinations are used foradditional experiments. The flux of gasses through the selected gaspermeable membranes is first quantified and modeled by abioticexperiments. The flux of O₂ from the vapor phase outside of the reactorinto the liquid phase uses the initial slope method and is measuredusing dissolved oxygen probes and medium that is initially anoxic. Theflux of carbon dioxide from a carbon dioxide saturated liquid phase intothe vapor phase also uses the initial slope method and is measured bytotal inorganic carbon analysis and with pH probes (a measure ofcarbonic acid). The dissolved inorganic carbon phase is 0%, 0.5% and 5%carbon dioxide initially. The data is integrated into the moving frontfungal growth models to develop more accurate parameters.

The best performing membrane/feedstock combinations observed are thenused for detailed biotic optimization experiments aided by a fungalgrowth model. Both glass trays and Petri dishes are used. The smallerPetri dishes facilitate the intensive destructive sampling for biomassand liquid analyses over time. Creation of conditions wherein nearly allof the added carbon and nutrients are converted into biomass withminimal wastes are identified. Here, carbon and electron fluxes andreactor conditions are evaluated by measuring the biomass produced perelectron donor and biomass produced per electron acceptor yields. Theelemental composition of the biomass is measured using commercialservices (e.g. Microanalysis Inc., Wilmington Del.) to complete the massbalances. Parameters of interest include volumes of the liquid phase andconcentrations of available feedstock and nutrients (carbon substrate,nitrogen source, inorganic nutrients, oxygen). The resulting data isused in a moving front mathematical model of fungal mat growth thatfacilitates a quantitative comparison and ultimately optimization ofgrowth conditions.

1. A food product, comprising: a piece of a filamentous fungal biomat, wherein the biomat consists essentially of mycelia and has been subjected to a treatment to eliminate cell viability; at least one additive selected from the group consisting of flavors, spices, flavor enhancers, fats, fat replacers, preservatives, sweeteners, color additives, nutrients, emulsifiers, stabilizers, thickeners, pH control agents, acidulants, leavening agents, anti-caking agents, humectants, yeast nutrients, dough strengtheners, dough conditioners, firming agents, enzyme preparations, gases, and combinations thereof; and wherein the piece is of a size consistent with the food product.
 2. The food product of claim 1, wherein the at least one additive is selected from the group consisting of flavors, spices, flavor enhancers, and sweeteners.
 3. The food product of claim 1, wherein the food product is a meat substitute.
 4. The food product of claim 1, wherein the food product is a foodstuff comparable to at least one of tofu, bacon, and jerky.
 5. The food product of claim 1, wherein the at least one additive comprises a spice mixture selected from the group consisting of a Cajun spice mixture, a cheese spice mixture, a soy spice mixture, a vinegar spice mixture, an herb spice mixture, a sour cream and onion spice mixture, a liquid smoke spice mixture, and a vegan meat spice mixture.
 6. The food product of claim 1, wherein the treatment to eliminate cell viability is selected from the group consisting of heating, irradiation, steaming, and combinations thereof.
 7. The food product of claim 1, wherein the piece of the filamentous fungal biomat has been size-reduced from the biomat by a means selected from the group consisting of cutting the biomat, chopping the biomat, dicing the biomat, mincing the biomat, grinding the biomat, blending the biomat, sonicating the biomat, and combinations thereof.
 8. The food product of claim 1, wherein the piece of the filamentous fungal biomat has been subjected to tenderization.
 9. The food product of claim 1, wherein the food product is prepared by contacting the piece of the filamentous fungal biomat with the at least one additive and dehydrating the biomat.
 10. The food product of claim 1, comprising all essential amino acids.
 11. The food product of claim 1, wherein the piece of the filamentous fungal biomat is the source for at least about 25 wt % of protein in the food product.
 12. The food product of claim 11, wherein the piece of the filamentous fungal biomat is the sole protein component in the food product.
 13. The food product of claim 1, wherein the piece of the filamentous fungal biomat comprises at least about 46 wt % protein.
 14. The food product of claim 1, wherein the food product is vegan.
 15. The food product of claim 1, wherein the piece of the filamentous fungal biomat is produced by surface fermentation.
 16. A food material, comprising filamentous fungal particles having an average particle size of about 75 to about 120 microns and a moisture content of less than about 14%, wherein the food material is a flour, wherein the filamentous fungal particles consist essentially of mycelia, and wherein the filamentous fungal particles have been subjected to a treatment to eliminate cell viability.
 17. The food material of claim 16, wherein at least about 5 wt % of the filamentous fungal particles have a particle size of at least about 180 microns and at least about 5 wt % of the filamentous fungal particles have a particle size of less than about 75 microns.
 18. The food material of claim 16, wherein the filamentous fungal particles have a moisture content of less than about 12%.
 19. The food material of claim 16, wherein the filamentous fungal particles have one or more of the following characteristics: comprise all essential amino acids; have a fiber content of at least about 23 wt %; and have a lipid content of about 6 wt % to about 11 wt %.
 20. The food material of claim 16, wherein the filamentous fungal particles are the source for at least about 25 wt % of protein in the food material.
 21. The food material of claim 16, wherein the filamentous fungal particles comprise at least about 46 wt % protein.
 22. The food material of claim 16, wherein the food material is vegan.
 23. A food material, comprising filamentous fungal particles having an average particle length of about 0.4 mm to about 500 mm, an average particle width of about 0.4 mm to about 7 mm, and an average particle height of about 0.03 mm to about 1.0 mm, wherein the filamentous fungal particles consist essentially of mycelia and have been subjected to a treatment to eliminate cell viability.
 24. The food material of claim 23, wherein the average particle length is about 4 mm to about 10 mm, the average particle width is about 1.0 mm to about 3 mm, and the average particle height is less than about 0.75 mm.
 25. The food material of claim 23, wherein the average particle length is less than about 1.5 mm, the average particle width is less than about 1 mm, and the average particle height is less than about 0.75 mm.
 26. The food material of claim 23, comprising all essential amino acids.
 27. The food material of claim 23, wherein the filamentous fungal particles are the source for at least about 25 wt % of protein in the food material.
 28. The food material of claim 23, wherein the filamentous fungal particles comprise at least about 46 wt % protein.
 29. The food material of claim 23, wherein the food material is vegan.
 30. A food product comprising the food material of claim 23 and selected from the group consisting of a meat product, a chicken breast, a chicken nugget, a ground chicken product, a fish ball, a fish filet, a fish patty, a fish stick, a jerky product, a pasta product, a noodle, a dumpling, a pie, a confection, and a candy. 